NOVEL MICROORGANISM STRAIN FOR HIGH-PERFORMANCE METABOLISM OF BIOMASS-DERIVED CARBON SOURCE

Abstract

The present invention relates to a novel microorganism capable of metabolizing various carbon sources at high rates. A novel microorganism according to the present invention was observed to grow at a very high rate in a minimal medium/nutrient medium, etc., compared to microorganisms such as Escherichia coli, and shows resistance at a high initial sugar/salt concentrations as well as being able to produce lycopene and 2,3-butanediol through genetic manipulation. Therefore, the novel microorganism can be used in various production fields of high value-added compounds using microorganisms.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is based on and claims priority from Korean Patent Application No. 10-2018-0054287, filed on May 11, 2018, with the Korean Intellectual Property Office, the disclosure of which is incorporated herein in its entirety by reference.

TECHNICAL FIELD

[0002] The present disclosure relates to a novel microorganism capable of metabolizing various carbon sources at high rates.

BACKGROUND

[0003] Due to various environmental problems such as depletion of petroleum resources and generation of greenhouse gases, there is a need for a technology for sustainable and eco-friendly production of many compounds previously produced from petrochemical processes. Biomass has been modified over time to fossil fuels as basis of a conventional industry. In order to replace the fossil fuels, the biomass as a carbon source must be converted efficiently.

[0004] In order to convert the biomass to a target compound, sugar contained in the biomass must be converted into a desired metabolite through a fermentation process of a microorganism. Until now, glucose obtained from starch crops such as corn and sugar cane has been used in the fermentation process. However, in a near future, when a demand for bio compound is soaring, it is expected that many problems will be caused by soaring grain prices. For this reason, efforts have been made to replace the raw materials needed for bioprocessing with natural-rich land and marine plants.

[0005] In particular, among various biomass existing in nature, brown macroalgae (Saccharina japonica, etc.) is attracting attention as a next-generation raw material. Brown microalgae have a higher carbon dioxide fixation rate than conventional land plants and have a fast growth rate to secure a large amount of biomass. Further, the fermentation process is not inhibited because there is no lignin, and an expensive pre-treatment process is not necessary. However, carbon source rich in the brown microalgae is alginic acid (a homopolymer of .alpha.-L-guluronate and .beta.-D-mannuronate). In this connection, industrial microbes do not contain alginic acid metabolic pathway and thus cannot carry out metabolism.

[0006] Therefore, it is necessary to secure microorganisms which are capable of metabolizing various carbon sources containing alginic acid at high rates. There is also a need for a technology that may convert the carbon source to high value compounds via appropriate genetic engineering improvements. Securing and developing the strains will greatly contribute to the sustainable production of various materials such as biofuels, platform compounds and pharmaceuticals from the various biomasses.

SUMMARY

[0007] The present inventors have completed the present disclosure by developing microorganisms that may quickly metabolize various carbon sources in order to produce compounds environmentally friendly that have been produced by conventional petrochemical processes.

[0008] The present disclosure has been made in an effort to provide a Vibrio sp. DHG strain having an accession number of KCTC13239BP with high capabilities to utilize various carbon sources.

[0009] The present disclosure has been made in an effort to provide a transformed Vibrio sp. DHG strain in which a gene encoding a gamma protein represented by a nucleotide sequence of SEQ ID NO: 6 is introduced into the DHG strain.

[0010] The present disclosure has been made in an effort to provide a transformed strain for lycopene production, in which a crtEBI gene represented by a nucleotide sequence of SEQ ID NO: 9 is introduced into the transformed DHG strain.

[0011] The present disclosure has been made in an effort to provide a transformed strain for producing 2,3-butanediol, in which one or more genes selected from the group consisting of a budA gene represented by a nucleotide sequence of SEQ ID NO: 13, a budB gene represented by a nucleotide sequence of SEQ ID NO: 14 and a budC gene represented by a nucleotide sequence of SEQ ID NO: 15 are introduced into the transformed DHG strain.

[0012] The present disclosure has been made in an effort to provide a method for producing lycopene, the method comprising culturing the transformed strain for lycopene production.

[0013] The present disclosure has been made in an effort to provide a method for producing 2,3-butanediol, the method comprising culturing the transformed strain for producing the 2,3-butanediol.

[0014] The present disclosure has been made in an effort to provide an SXT recombinant system expression cassette comprising a synthetic 5' UTR (untranslated region), a promoter and a target gene, a flippase gene expression cassette, a crtEBI gene expression cassette or a budACB operon expression cassette.

[0015] The present disclosure has been made in an effort to provide a recombinant vector comprising the SXT recombination system expression cassette, flippase gene expression cassette, crtEBI gene expression cassette or budACB operon expression cassette.

[0016] An exemplary embodiment of the present disclosure provides a Vibrio sp. DHG strain having an accession number of KCTC13239BP with a carbon source high-performance metabolic pathway.

[0017] Another exemplary embodiment of the present disclosure provides a transformed Vibrio sp. DHG strain in which a gene encoding a gamma protein represented by a nucleotide sequence of SEQ ID NO: 6 is introduced into the DHG strain.

[0018] Yet another exemplary embodiment of the present disclosure provides a transformed strain for lycopene production, in which a crtEBI gene represented by a nucleotide sequence of SEQ ID NO: 9 is introduced into the transformed DHG strain.

[0019] Yet another exemplary embodiment of the present disclosure provides a transformed strain for producing 2,3-butanediol, in which one or more genes selected from the group consisting of a budA gene represented by a nucleotide sequence of SEQ ID NO: 13, a budB gene represented by a nucleotide sequence of SEQ ID NO: 14 and a budC gene represented by a nucleotide sequence of SEQ ID NO: 15 are introduced into the transformed DHG strain.

[0020] Yet another exemplary embodiment of the present disclosure provides a method for producing lycopene, the method comprising culturing the transformed strain for lycopene production.

[0021] Yet another exemplary embodiment of the present disclosure provides a method for producing 2,3-butanediol, the method comprising culturing the transformed strain for producing the 2,3-butanediol.

[0022] Yet another exemplary embodiment of the present disclosure provides an SXT recombinant system expression cassette comprising a synthetic 5' UTR (untranslated region), a promoter represented by one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 22 to 35 and 56, and one or more genes selected from the group consisting of genes encoding beta, exo and gamma proteins.

[0023] Yet another exemplary embodiment of the present disclosure provides a recombinant vector comprising the SXT recombinant system expression cassette.

[0024] Yet another exemplary embodiment of the present disclosure provides a flippase gene expression cassette comprising a synthetic 5' UTR (untranslated region), a promoter represented by one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 22 to 35 and 58, and a gene coding flippase.

[0025] Yet another exemplary embodiment of the present disclosure provides a recombinant vector comprising the flippase gene expression cassette.

[0026] Yet another exemplary embodiment of the present disclosure provides a crtEBI gene expression cassette comprising a synthetic 5' UTR (untranslated region), a promoter represented by one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 22 to 35 and 60, and a crtEBI gene.

[0027] Yet another exemplary embodiment of the present disclosure provides a recombinant vector comprising the crtEBI gene expression cassette.

[0028] Yet another exemplary embodiment of the present disclosure provides a budACB operon expression cassette comprising a synthetic 5' UTR (untranslated region), a promoter represented by one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 22 to 35 and 68, and at least one gene selected from the group consisting of genes coding BudA, BudC and BudB proteins.

[0029] Yet another exemplary embodiment of the present disclosure provides a recombinant vector comprising the BudACB operon expression cassette.

[0030] According to the exemplary embodiments of the present disclosure, the Vibrio sp. DHG strain according to the present invention was observed to grow at a very high rate in a minimal medium/nutrient medium, etc., compared to microorganisms such as Escherichia coli, and shows resistance at a high initial sugar/salt concentrations as well as being able to produce lycopene and 2,3-butanediol through genetic manipulation. Therefore, the Vibrio sp. DHG strain can be used in various production fields of high value-added compounds using microorganisms.

[0031] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG. 1 shows a growth rate of a Vibrio sp. DHG strain based on a carbon source according to the present disclosure.

[0033] FIG. 2 shows a resistance of a Vibrio sp. DHG strain to an initial sugar concentration according to the present disclosure.

[0034] FIG. 3 shows a resistance of a Vibrio sp. DHG strain to a salt concentration in the medium to according to the present disclosure.

[0035] FIG. 4 shows a result of checking whether plasmid pACYC or pUC are introduced into a Vibrio sp. DHG strain via colony PCR according to the present disclosure.

[0036] FIG. 5 shows a result of checking whether plasmid pACYC or pUC are introduced into a Vibrio sp. DHG strain via colony PCR according to the present disclosure.

[0037] FIG. 6 shows a result of checking a fluorescent expression when transforming the Vibrio sp. DHG strain according to the present disclosure with a fluorescent protein expression plasmid as a heterologous protein.

[0038] FIG. 7 shows a result of checking a degree of fluorescence expression of the transformed strain based on a synthetic promoter sequence.

[0039] FIG. 8 shows a result of checking a degree of fluorescence expression of the transformed strain based on a 5' UTR sequence.

[0040] FIG. 9 shows a cleavage map of a plasmid pACYCA_SXT according to the present disclosure for gamma protein expression.

[0041] FIG. 10 shows a cleavage map of a plasmid pRSF_FLP comprising an antibiotic resistance gene according to the present disclosure.

[0042] FIG. 11 shows a schematic diagram of a genetic manipulation method of a Vibrio sp. DHG strain according to the present disclosure.

[0043] FIG. 12 shows a structure and targeting site of a double stranded DNA used for genetic manipulation of a Vibrio sp. DHG strain according to the present disclosure.

[0044] FIG. 13 shows a result of measuring a lycopene production amount by the transformed strain for lycopene production obtained by transforming the Vibrio sp. DHG strain according to the present disclosure.

[0045] FIG. 14 shows a map of a plasmid pACYC_BudACB for 2,3-butanediol production according to the present disclosure.

[0046] FIG. 15 shows a result of identifying gene deletion of the transformed strain for producing 2,3-butanediol according to the present disclosure through colony PCR.

[0047] FIG. 16 shows a result of a metabolite of the transformed strain for 2,3-butanediol production according to the present disclosure and measuring a 2,3-butanediol production amount by the transformed strain.

[0048] FIG. 17 shows a map of a plasmid pACYC_idi_ispA_crtEBI for lycopene production according to the present disclosure.

[0049] FIG. 18 shows a map of a plasmid pACYC_idi_ispA_crtEBI_dxs for lycopene production according to the present disclosure.

DETAILED DESCRIPTION

[0050] In the following detailed description, reference is made to the accompanying drawing, which forms a part hereof. The illustrative embodiments described in the detailed description, drawing, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented here.

[0051] Hereinafter, the present disclosure will be described in detail.

[0052] Unless otherwise defined, all terms including technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this inventive concept belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0053] In the following detailed description of the present disclosure, numerous specific details are set forth in order to provide a thorough understanding of the present disclosure. However, it will be understood that the present disclosure may be practiced without these specific details. In other instances, well-known methods, procedures, components, and circuits have not been described in detail so as not to unnecessarily obscure aspects of the present disclosure.

[0054] According to an aspect of the present disclosure, the present disclosure provides a Vibrio sp. DHG strain having an accession number of KCTC13239BP with a carbon source high-performance metabolic pathway.

[0055] As used herein, the term "carbon source" refers to a carbon compound that is assimilated by a living body and used as a bio-constituent carbon. In culturing strains, carbon sources are used to identify physiological relationships between nutrients and the strains such that isolation and growth characteristics of the strains are identified.

[0056] The carbon source may be sugar or sugar alcohol. More specifically, the carbon source may include one or more selected from the group consisting of glucose, mannitol, sucrose, arabinose, galactose, glycerol, xylose, mannose, fructose, lactose, maltose, sucrose, alginic acid, cellulose, dextrin, glycogen, hyaluronic acid, lentinan, Zymosan, chitosan, glucan, lignin, and pectin. Preferably, the carbon source may include one or more selected from the group consisting of glucose, mannitol, alginic acid, sucrose, arabinose, galactose and glycerol, but is not limited thereto.

[0057] As used herein, the term "carbon source high-performance metabolic pathway" refers to a metabolic pathway containing enzymes that may metabolize various sugars or sugar alcohols. Microorganisms having the carbon source high-performance metabolic pathway may use a mixed sugar containing one or more sugars or sugar alcohols as the carbon source.

[0058] In one embodiment of the present disclosure, the strain includes a 16S rDNA gene represented by a nucleotide sequence of a SEQ ID NO: 1.

[0059] As used herein, the term "gene" should be considered in the broadest sense, and may encode a structural or regulatory protein. In this connection, the regulatory protein includes a transcription factor, a heat shock protein or a protein involved in DNA/RNA replication, transcription and/or translation. In the present disclosure, a target gene that is subject to expression inhibition may be present as an extrachromosomal component.

[0060] In another embodiment of the present disclosure, the strain comprises an SXT recombination system. More specifically, the strain may comprise a beta gene represented by a nucleotide sequence of SEQ ID NO: 2 or a beta protein represented by an amino acid sequence of SEQ ID NO: 3 and an exo gene represented by a nucleotide sequence of SEQ ID NO: 4 or an exo protein represented by an amino acid sequence of SEQ ID NO: 5. Further, the strain may comprise functional equivalents of the genes or proteins described above. The term "functional equivalent" means polynucleotides having at least 70%, preferably at least 80%, more preferably at least 90%, more preferably at least 95% sequence homology with the nucleotide sequence of the beta or exo gene via deletion, substitution or insertion of the nucleotide. The functional equivalent refers to a polynucleotide that exhibits substantially homogeneous physiological activity with the genes described above. The "% sequence homology" of the polynucleotide is identified by comparing a comparison region with two optimally arranged sequences. A portion of the polynucleotide sequence in the comparison region may include an addition or deletion (that is, gap) compared to a reference sequence (not including an additions or deletion) for the optimal alignment of the two sequences.

[0061] Further, the functional equivalents have at least 80% or more, preferably 90%, more preferably 95% or more sequence homology (i.e., identity) with the beta or exo proteins described above via the addition, substitution or deletion of amino acids. More preferably, the functional equivalents have, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology with the beta or exo proteins via the addition, substitution or deletion of amino acids. The functional equivalents refer to a peptide that exhibits substantially the same physiological activity as the protein described above. As used herein, sequence homology and homogeneity are defined as the percentage of amino acid residues in the candidate sequence relative to the amino acid sequence of the aforementioned protein after aligning the amino acid sequence and the candidate sequence of the aforementioned protein and introducing gaps. If necessary, conservative substitutions as part of sequence homogeneity are not considered in order to obtain maximum percentage sequence homogeneity. N-terminal, C-terminal or internal elongation, deletion or insertion of the amino acid sequence of the aforementioned protein is not to be construed as a sequence affecting sequence homology or homology. Further, the sequence homogeneity may be determined by common standard methods used to compare similar portions of the amino acid sequences of two polypeptides. Computer programs such as BLAST or FASTA align the two polypeptides so that their respective amino acids are optimally matched with each other (in accordance with the full length sequence of one or two sequences or the predicted portion of one or two sequences). The program provides a default opening penalty and default gap penalty, and provides scoring metrics such as PAM250 (Standard Scoring Matrix; Dayhoff et al., in Atlas of Protein Sequence and Structure, vol 5, supp. 3, 1978) as used in conjunction with a computer program. For example, the percentage homogeneity may be calculated as follows: The total number of identical matches is multiplied by 100 and then the result is divided by the sum of the length of the longer sequence in the matched span and the number of gaps introduced into the longer sequence to align the two sequences.

[0062] The present inventors isolated and identified the Vibrio sp. DHG strain from sea microalgae sludge and deposited the Vibrio sp. DHG strain with the Korea Institute of Biotechnology and Biotechnology Center on Apr. 6, 2017 and received the accession number KCTC13239BP. The Vibrio sp. DHG strain has a very high growth rate in the minimum medium and nutrient medium compared to microorganisms such as Escherichia coli and is resistant to high initial sugar/salt concentrations.

[0063] Another aspect of the present disclosure provides a transformed Vibrio sp. DHG strain in which a gene encoding a gamma protein represented by a nucleotide sequence of SEQ ID NO: 6 is introduced into the Vibrio sp. DHG strain.

[0064] In a preferred embodiment of the present disclosure, the gamma gene is preferably introduced into the Vibrio sp. DHG strain using the vector pACYCA_SXT shown in FIG. 9.

[0065] Since the transformed DHG strain in accordance with the present disclosure comprises the SXT recombination system, the strain may be transformed using a plasmid as it is for improving E. coli, which has been conventionally used. Through the transformation, the strain can be produced capable of producing the high value-added compounds.

[0066] Yet another aspect of the present disclosure provides a transformed strain for lycopene production, in which a crtEBI gene represented by a nucleotide sequence of SEQ ID NO: 9 is introduced into the transformed DHG strain.

[0067] In one embodiment of the present disclosure, the transformed strain for lycopene production may be obtained by additionally introducing the idi gene represented by the nucleotide sequence of SEQ ID NO: 10 into the transformed DHG strain. In addition, the transformed strain for lycopene production may be obtained by additionally introducing the ispA gene represented by the nucleotide sequence of SEQ ID NO: 11 into the transformed DHG strain. The idi and ispA genes may be introduced simultaneously with the crtEBI gene or may be introduced sequentially therewith. In one embodiment of the present disclosure, the crtEBI, idi and ispA genes were introduced into the transformed DHG strain using the vector pACYC_idi_ispA_crtEBI described in FIG. 17.

[0068] In another embodiment of the present disclosure, the transformed strain for lycopene production may be obtained by additionally introducing the dxs gene represented by the nucleotide sequence of SEQ ID NO: 12 into the transformed DHG strain. Further, the dxs gene may be introduced simultaneously or sequentially with the crtEBI gene. The idi and/or ispA genes may be introduced simultaneously or sequentially with the crtEBI gene. However, the present disclosure is not limited thereto.

[0069] In one embodiment of the present disclosure, the crtEBI, dxs, idi and ispA genes were introduced simultaneously into the transformed DHG strain. The genes were introduced into the transformed DHG strain using the vector pACYC_idi_ispA_crtEBI_dxs represented by the cleavage map shown in FIG. 18.

[0070] Yet another aspect of the present disclosure provides a transformed strain for producing 2,3-butanediol, in which one or more genes selected from the group consisting of a budA gene represented by a nucleotide sequence of SEQ ID NO: 13, a budB gene represented by a nucleotide sequence of SEQ ID NO: 14 and a budC gene represented by a nucleotide sequence of SEQ ID NO: 15 are introduced into the transformed DHG strain.

[0071] In one embodiment of the present disclosure, a budACB operon composed of the budA, budB and budC genes was introduced into the transformed DHG strain using a vector pACYC_BudACB represented by the cleavage map shown in FIG. 14.

[0072] In another embodiment of the present disclosure, in order to improve the productivity and yield of 2,3-butanediol, the transformed strain for producing 2,3-butanediol may be obtained by deleting one or more genes selected from the group consisting of the ldhA gene encoding the enzyme producing lactic acid, frdABCD operon encoding enzymes producing succinic acid and pflB gene encoding an enzyme converting pyruvate to acetyl-CoA from the Vibrio sp. DHG strain. More specifically, the transformed strain for producing 2,3-butanediol may be obtained by deleting one or more genes selected from the group consisting of ldhA gene represented by the nucleotide sequence of SEQ ID NO: 16, frdA gene represented by the nucleotide sequence of SEQ ID NO: 17, frdB gene represented by the nucleotide sequence of SEQ ID NO: 18, frdC gene represented by the nucleotide sequence of SEQ ID NO: 19, frdD gene represented by the nucleotide sequence of SEQ ID NO: 20, and pflB gene represented by the nucleotide sequence of SEQ ID NO: 21 from the DHG strain. The gene deletion may use pRSF_FLP as a vector represented by the cleavage map shown in FIG. 10. The present disclosure is not limited thereto.

[0073] Yet another aspect of the present disclosure provides a method for producing lycopene, the method comprising culturing the transformed strain for lycopene production.

[0074] Yet another aspect of the present disclosure provides a method for producing 2,3-butanediol, the method comprising culturing the transformed strain for producing the 2,3-butanediol.

[0075] The medium and other culture conditions used for the cultivation of the microorganisms in accordance with the present disclosure may be any medium used for the cultivation of microorganisms of the Vibrio sp. DHG. However, the requirements of the microorganisms in accordance with the present disclosure should be satisfactorily met. Preferably, the microorganism in accordance with the present disclosure may be incubated in a conventional medium containing a suitable carbon source, nitrogen source, amino acids, vitamins and the like under aerobic conditions while controlling the temperature, pH and the like.

[0076] In a preferred embodiment of the present disclosure, the medium may contain sugar or sugar alcohol as a carbon source. More specifically, the medium may contain at least one selected from the group consisting of glucose, mannitol, sucrose, arabinose, galactose, glycerol, xylose, mannose, fructose, lactose, maltose, sucrose, alginic acid, cellulose, dextrin, glycogen, hyaluronic acid, lentinan, Zymosan, chitosan, glucan, lignin and pectin. Preferably, the medium may contain at least one selected from the group consisting of glucose, mannitol, alginic acid, sucrose, arabinose, galactose and glycerol. However, the present disclosure is not limited thereto. The inorganic compound in the medium may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate and calcium carbonate. In addition, amino acids, vitamins and appropriate precursors may be contained in the medium. These media or precursors may be added batchwise or continuously to the culture.

[0077] During the culture, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid may be added to the culture in an appropriate manner to adjust the pH of the culture. Further, during culturing, antifoaming agents such as fatty acid polyglycol esters may be used to inhibit bubble generation. Further, in order to maintain the aerobic state of the culture, oxygen or oxygen containing gas may be injected into the culture, and in order to maintain anaerobic and aerobic states, gas may not be injected or nitrogen, hydrogen or carbon dioxide gas may be injected.

[0078] The temperature of the culture may usually be set to 27.degree. C. to 37.degree. C., preferably 30.degree. C. to 35.degree. C. The incubation period may continue until the desired amount of useful substance is obtained. Preferably, the cell may be incubated for 10 to 100 hours.

[0079] The compound produced at the culturing stage in accordance with the present disclosure (lycopene or 2,3-butanediol) may be further subjected to a purification or obtaining step. A method for obtaining itaconic acid from microorganisms or cultures may be used by methods known in the art, such as centrifugation, filtration, anion exchange chromatography, crystallization and HPLC, but are not limited to these examples.

[0080] The obtaining step may include a purification process. Those skilled in the art may select and utilize one as needed from a variety of known purification processes.

[0081] Yet another aspect of the present disclosure provides provide an expression cassette comprising a synthetic 5' UTR (untranslated region), a promoter and a target gene.

[0082] One embodiment of the present disclosure provides an SXT recombinant system expression cassette comprising a synthetic 5' UTR (untranslated region), a promoter and a target gene, a flippase gene expression cassette, a crtEBI gene expression cassette or a budACB operon expression cassette.

[0083] One embodiment of the present disclosure provides an SXT recombinant system expression cassette comprising a synthetic 5' UTR (untranslated region), a promoter represented by one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 22 to 35 and 56, and one or more genes selected from the group consisting of genes encoding beta, exo and gamma proteins. More specifically, the synthetic 5' UTR is preferably represented by the nucleotide sequence of SEQ ID NO: 57.

[0084] One embodiment of the present disclosure provides a flippase gene expression cassette comprising a synthetic 5' UTR (untranslated region), a promoter represented by one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 22 to 35 and 58, and a gene encoding flippase. More specifically, the synthetic 5' UTR is preferably represented by a nucleotide sequence of SEQ ID NO: 59. Further, the flippase gene expression cassette may further comprise an idi gene expression cassette or an ispA gene expression cassette. The idi gene expression cassette preferably comprises a synthetic 5' UTR represented by the nucleotide sequence of SEQ ID NO: 65, a promoter represented by one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 22 to 35 and 64, and an ispA gene. Further, the ispA gene expression cassette may preferably comprise a synthetic 5' UTR represented by the nucleotide sequence of SEQ ID NO: 67, a promoter represented by one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 22 to 35 and 66, and a dxs gene.

[0085] One embodiment of the present disclosure provides a crtEBI gene expression cassette comprising a synthetic 5' UTR (untranslated region), a promoter represented by one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 22 to 35 and 60, and a crtEBI gene. In this connection, the synthetic 5' UTR is preferably represented by the nucleotide sequence of SEQ ID NO: 61.

[0086] One embodiment of the present disclosure provides a budACB operon expression cassette comprising a synthetic 5' UTR (untranslated region), a promoter represented by one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 22 to 35 and 68, and at least one gene selected from the group consisting of genes coding BudA, BudC and BudB proteins.

[0087] As used herein, the term "5' UTR (untranslated region)" refers to an untranslated region at a 5' terminal and 3' terminal of the mRNA. Typically, the 5' untranslated region (5' UTR) performs several functions in the gene expression process, the most important of which involves in the regulation of mRNA translation efficiency. The nucleotide sequence of the 5' UTR at an adjacent upper portion of the translation initiation codon has been reported to affect the efficiency of the translation step. The 5' UTR is composed of nucleotide of 100 bases or more in length. The 3' UTR has a length of a few kilo bases longer than the 5' UTR. Further, the results of studies reported that eukaryotes have a ribosome binding site sequence as a sequence belonging to the 5' UTR which is not a fixed position such as the Shine-Dalgarno sequence which is known as a ribosome binding site sequence located in the 5' UTR in prokaryotes.

[0088] As used herein, the term "expression cassette" refers to a unit cassette that contains a promoter and a gene encoding a target protein and may be expressed to produce a target protein operably linked to a downstream of the promoter. Inside or outside of the expression cassette, a variety of factors that may help the efficient production of the protein of interest may be contained. In the target protein expression cassette, a gene encoding the target protein may be specifically operably linked to a downstream of the promoter sequence.

[0089] The "operably linked" means that the gene sequence and the promoter sequence are functionally linked to each other so that the nucleic acid sequence having the promoter activity according to the present disclosure initiates and mediates the transcription of the gene encoding the protein of interest. The operable linkages may be made using genetic recombination techniques known in the art. Site-specific DNA cleavage and ligation may be made using, but are not limited to, cleavage and ligation enzymes in the art.

[0090] As used herein, the term "target protein" refers to a protein to be expressed from a microorganism. Specifically, the target protein may include any protein to be expressed from a recombinant microorganism without restriction. Examples thereof include, but are not limited to, proteins, flippase, lycopene producing enzyme, and BudACB constituting the SXT recombination system.

[0091] The recombinant gene expression cassette may be inserted into chromosome of a host cell to prepare recombinant microorganisms. For those skilled in the art to which the present disclosure belongs, it is obvious that the insertion of the recombinant gene expression cassette into the genomic chromosome of the host cell will have the same effect from the introduction of the recombinant vector into the host cell.

[0092] The recombinant gene expression cassette may be inserted into chromosome of a host cell to prepare recombinant microorganisms. For those skilled in the art to which the present disclosure belongs, it is obvious that the insertion of the recombinant gene expression cassette into the genomic chromosome of the host cell will have the same effect from the introduction of the recombinant vector into the host cell.

[0093] A method of inserting the recombinant gene expression cassette into a chromosome of a host cell may include a commonly known gene engineering method. One example thereof is a method using a retroviral vector, adenovirus vector, adeno-associated virus vector, herpes simplex virus vector, poxvirus vector, lentiviral vector or non-viral vector.

[0094] As used herein, the term "promoter" refers to a non-translated nucleic acid sequence of an upstream of a coding region, containing a binding site to polymerase and having transcription initiation activity of a promoter downstream gene to mRNA, that is, a DNA region that binds to polymerase to allow initiation of transcription of a gene. The promoter may be located at a 5' site of the mRNA transcription initiation site.

[0095] The promoter nucleic acid molecule according to the present disclosure may be isolated or prepared using standard molecular biology techniques. For example, the promoter nucleic acid molecule according to the present disclosure may be prepared using standard synthesis techniques using an automated DNA synthesizer. However, the present disclosure is not limited thereto.

[0096] In accordance with the present disclosure, the promoters may result in expression of a target gene operably linked to a nucleic acid molecule having the promoter activity in a desired microorganism.

[0097] Further, the promoter sequence according to the present disclosure may be easily modified by those skilled in the art by conventionally known mutagenesis, such as directional evolution and site-specific mutagenesis. Thus, the promoter may include nucleotide sequences having homology of at least 70%, specifically at least 80%, more specifically at least 90%, more specifically at least 95%, even more specifically at least 98%, and most specifically, at least 99% with the nucleotide sequence of the SEQ ID NOs: 22 to 35, without any limitation. Further, any nucleotide sequence obtained by deletion, modification, substitution, or addition of a portion of the nucleotide sequence having the above ranged homology and having the promoter activity should be interpreted to fall within the scope of the present disclosure.

[0098] As used herein, the term "homology" refers to a percentage of identity between two polynucleotide or polypeptide moieties. Homology between sequences from one moiety to another may be determined by known techniques. For example, the homology may be determined by directly aligning sequence information between two polynucleotide molecules or two polypeptide molecules, such as parameters including scores, identities, and similarities, or aligning the sequence information using a computer program readily available (e.g., BLAST 2.0). Further, the homology between polynucleotides may be determined by hybridization of polynucleotides under conditions of stable double stranding between homologous regions, followed by decomposition thereof with single-strand-specific nucleases to determine a size of the decomposed fragment.

[0099] According to another aspect according to the present disclosure, there is provided a recombinant vector comprising the SXT recombinant system expression cassette, flippase gene expression cassette, crtEBI gene expression cassette or BudACB operon expression cassette.

[0100] As used herein, the term "vector" refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA in a suitable host. The vector may be plasmids, phage particles or simply potential genomic inserts. Once the vector has been transformed into a suitable host, the vector may replicate and function independently of the host genome, or in some cases may be integrated into the genome itself. Since plasmids are the most commonly currently used form of the vector, the "plasmid" and "vector" are sometimes used interchangeably in the present disclosure. For purposes of the present disclosure, it is preferable to use a plasmid vector. Typical plasmid vectors that may be used for this purpose may have a structure including (a) a replication initiation point that allows for efficient replication to contain several to several hundred plasmid vectors per host cell, (b) an antibiotic resistance gene that allows selection of a host cell transformed with a plasmid vector, and (c) a restriction enzyme cleavage site into which foreign DNA fragments may be inserted. Although no suitable restriction enzyme cleavage site is present, synthetic oligonucleotide adapters or linkers according to conventional methods may be used to facilitate ligation of the vector and foreign DNA. After the ligation, the vector should be transformed into the appropriate host cell. The transformation may be easily accomplished using calcium chloride method or electroporation or the like.

[0101] As is well known in the art, in order to raise the expression level of a transfected gene in a host cell, the gene must be operably linked to transcriptional and translational expression control sequences that function in the selected expression host. Preferably, the expression control sequence and the corresponding gene are contained in one recombinant vector containing the bacterial selection marker and the replication start point.

[0102] As used herein, the "recombinant vector" refers to a recombinant DNA molecule containing the desired coding sequence and the appropriate nucleic acid sequence necessary to express the coding sequence operably linked in a specific host organism. The recombinant vectors may preferably contain one or more selectable markers. The marker is typically a nucleic acid sequence having properties that may be selected by chemical methods, such as all genes that may distinguish transformed cells from non-transformed cells. Examples thereof include, but are not limited to, antibiotic resistance genes such as ampicillin, kanamycin, G418, bleomycin, hygromycin, chloramphenicol, and the like. The present disclosure may not be limited thereto. The maker may be suitably selected by those skilled in the art.

BEST MODE

[0103] Hereinafter, the present disclosure will be described in more detail with examples. These examples are intended only to illustrate the present disclosure. It will be apparent to those of ordinary skill in the art that the scope of the present disclosure is not to be construed as being limited by these examples.

Example 1. Isolation and Identification of Vibrio sp. DHG Strain from Sea Microalgae Sludge

[0104] In order to secure microorganisms that may grow rapidly in high concentration salts, samples were taken from seashore microalgae sludge and cultured in a laboratory.

[0105] In this connection, a composition of the medium as used is as follows.

[0106] NaCl 30 g/L

[0107] (NH.sub.4).sub.2SO.sub.4 5 g/L

[0108] K.sub.2HPO.sub.4 2 g/L

[0109] MgSO.sub.47H.sub.2O 0.5 g/L

[0110] Alginate 10 g/L

[0111] ATCC Trace mineral solution 2 ml/L

[0112] It was identified that the incubation of the samples in the medium leads to rapid growth thereof. Then, a single colony was separated.

[0113] 16S rDNA sequence (SEQ ID NO: 1) was analyzed to determine the species specificity of the isolated microorganism, and found to belong to Vibrio sp.

[0114] The present inventors named the microorganism as the Vibrio sp. DHG, and then deposited at the Korea Institute of Bioscience and Biotechnology on Apr. 6, 2017. The accession number KCTC13239BP was allocated thereto.

Example 2. Metabolic Carbon Source Analysis of Vibrio sp. DHG and Measurement of Growth Rate Thereof

[0115] Vibrio sp. DHG was cultured in a minimal medium with various carbon sources as the only carbon source in order to determine the type of a carbon source that the Vibrio sp. DHG can metabolize and the corresponding growth rate thereof (30.degree. C., 250 rpm).

[0116] A detailed medium composition for culturing the microorganism is as follows.

[0117] NaCl 30 g/L

[0118] (NH.sub.4)2SO.sub.4 5 g/L

[0119] K.sub.2HPO.sub.4 2 g/L

[0120] MgSO.sub.47H.sub.2O 0.5 g/L

[0121] Carbon source 10 g/L

[0122] ATCC Trace mineral solution 2 ml/L

[0123] The specific growth rate of the Vibrio sp. DHG strain is shown in FIG. 1.

[0124] As shown in FIG. 1, it may be identified that when considering that the glucose metabolism rate by E. coli is 0.5 to 0.6, the Vibrio sp. DHG strain is able to use all the carbon sources in the comparative bacteria as the only carbon source and grows at a high rate (>0.8 h.sup.-1). In addition, the carbon source metabolism per cell of the Vibrio sp. DHG strain was similar to or higher than the metabolism of glucose by E. coli. The result indicates that the strain can convert carbon sources quickly in biorefinery processes.

Example 3. Resistance Test to Initial Sugar (Substrate) Concentration

[0125] In order to identify the growth rate according to the initial sugar concentration in the incubator, we tried to test the resistance to glucose which exhibited the highest growth rate in the previous experiment. To do this, we added 20 mL of medium with different initial concentrations to the flask of a 350 mL, and added the Vibrio sp. DHG thereto at OD.sub.600 of 0.05. Then, the initial growth rates thereof were compared with each other. FIG. 2 shows a result of identifying the growth rate based on the initial sugar concentration.

[0126] As shown in FIG. 2, the Vibrio sp. DHG maintained a high growth rate at an initial substrate concentration of about 100 g/L.

Example 4. Resistance Test to Salt Concentration in Medium

[0127] To test the resistance of the Vibrio sp. DHG to salts in the medium, we compared the growth rates based on concentrations of salts that may be contained during fermentation. To do this, we added 20 mL of the medium with different initial concentrations to the flask of a 350 mL and added the Vibrio sp. DHG thereto at OD.sub.600 of 0.05. Then, the initial growth rates thereof were compared with each other. The result is shown in FIG. 3.

[0128] As shown in FIG. 3, the Vibrio sp. DHG was identified to grow in the presence of 10 to 40 g/L NaCl, 10 to 60 g/L Na.sub.2SO.sub.4 and about 10 to 100 g/L NaH.sub.2PO.sub.4. In conclusion, the Vibrio sp. DHG was identified to have high resistance to the salt.

Example 5. Plasmid Introduction for Transformation of Vibrio sp. DHG Strain

[0129] In general, the most basic method for transforming microorganisms is to introduce plasmids thereto. For efficient transformation, it is important to introduce plasmids commonly used in microorganisms such as E. coli.

[0130] In order to identify that the plasmid is introduced into the Vibrio sp. DHG strain, the following experiment was performed with reference to the transforming method of Vibrio natriegens.

[0131] (1) First, a seed cultured overnight in a brain heat infusion (BD) medium was inoculated to a fresh medium at a ratio of 1/100, and then incubated at 37.degree. C. at 200 rpm until the OD reaches 0.6.

[0132] (2) When the OD reached 0.6, the flask was placed on ice for 20 minutes and centrifuged at 4500 rpm for about 15 minutes to collect cells.

[0133] (3) After the cells were collected, 10 mL of sterile electroporation buffer (680 mM sucrose, 7 mM K.sub.2HPO.sub.4, pH 7) was added thereto, and then the cell pellet was resuspended, washed, and centrifuged again at 4500 rpm.

[0134] (4) This process was repeated four times.

[0135] (5) Finally, an appropriate amount of electroporation buffer was added thereto to resuspend the cells to adjust the OD to 16.

[0136] (6) After adding 500 ng or more of the plasmid to be used for transformation thereto, electroporation was performed with an electric shock of 0.8 kV.

[0137] In order to identify the transformation of the cells, we overnight cultured the cells in a plate containing an appropriate amount of antibiotics, and performed colony PCR to identify the presence of the plasmid in the microorganisms. The results are shown in FIG. 4.

[0138] As shown in FIG. 4, it was confirmed that plasmids pACYC and pUC were introduced thereto respectively.

[0139] Further, to further identify that two or more plasmids may be introduced into the microorganism in the same manner, the plasmids pACYC and pUC were transformed sequentially. As shown in FIG. 5, it was identified that, after liquid culturing the plasmid in a BHI medium and then purifying the plasmid, the two plasmids coexist and are secured when being electrophoresed.

Example 6. Heterologous Protein Expression in Vibrio sp. DHG Strain

[0140] The fast growth rate of the Vibrio sp. DHG suggests that the desired protein can be produced at a high speed. To this end, we identified the expression of heterologous proteins in the Vibrio sp. DHG strain.

[0141] Specifically, using the transforming method of Example 5, plasmids capable of expressing heterologous proteins were respectively introduced thereto. The plasmid capable of expressing the heterologous protein is designed such that the fluorescent protein GFP may be expressed under different promoters (PJ23100, Plac, Ptac, PT7, Ptet, Para). The experimental group transformed with each plasmid was cultured in nutrient medium (LBv2). Then, we identified whether the fluorescent protein GFP was produced by the experimental group. The results of identifying the heterologous protein production by the Vibrio sp. DHG strain are shown in FIG. 6.

[0142] As shown in FIG. 6, the Vibrio sp. DHG strain was able to produce heterologous proteins under various promoters.

Example 7. Quantitative Regulation of Transcription Through Constant Promoter Sequences

[0143] For the redesign of microorganisms, it is very important to quantitatively control the expression level of genes. This may be effectively applied to increasing of biochemical production, such as expansion of metabolic pathways through efficient overexpression of genes, and optimization of carbon flow through optimal expression. In general, such expression control has been largely performed in the transcription and translation stages of gene expression. The amount of expression in the transcription step depends on the sequence of the promoter that determines the affinity with the RNA polymerase. The sequence of -35 and -10 regions among the promoter sequences is known to be very important.

[0144] In order to regulate transcription in the Vibrio sp. DHG strain, the sequence of the promoter was randomly changed and then the expression level of sGFP was measured to identify the change in the transcription level. Specifically, a random sequence was placed in the -35 and -10 regions using the constant promoter J23100 provided from Partsregistry as a template. After linking the random sequence thereto to express the GFP gene, the plasmids having various sequence libraries were prepared. The promoters using the promoter J23100 as the template are shown in Table 1.

TABLE-US-00001 TABLE 1 Standardized relative Samples Sequence(5'->3') intensity SEQ ID NO Templates NNNNNNGCTAGCTCAGTCCTAGGKANNNNGCTAGC SEQ ID NO: 22 1 CTTATGGCTAGCTCAGTCCTAGGGACAGTGCTAGC 0.053 SEQ ID NO: 23 2 TTTACGGCTAGCTCAGTCCTAGGGATAGTGCTAGC 0.098 SEQ ID NO: 24 3 CTGACGGCTAGCTCAGTCCTAGGGATAGTGCTAGC 0.143 SEQ ID NO: 25 4 TTGATGGCTAGCTCAGTCCTAGGGATTATGCTAGC 0.171 SEQ ID NO: 26 5 TTGATGGCTAGCTCAGTCCTAGGTACAGTGCTAGC 0.254 SEQ ID NO: 27 6 TTGATGGCTAGCTCAGTCCTAGGTATTGTGCTAGC 0.288 SEQ ID NO: 28 7 TTGATGGCTAGCTCAGTCCTAGGTACTATGCTAGC 0.322 SEQ ID NO: 29 8 TTGACGGCTAGCTCAGTCCTAGGTACTGTGCTAGC 0.420 SEQ ID NO: 30 9 TTGATGGCTAGCTCAGTCCTAGGTACAATGCTAGC 0.514 SEQ ID NO: 31 10 TTGATGGCTAGCTCAGTCCTAGGTATAGTGCTAGC 0.579 SEQ ID NO: 32 11 TTGACGGCTAGCTCAGTCCTAGGTATTGTGCTAGC 0.651 SEQ ID NO: 33 12 TTGATGGCTAGCTCAGTCCTAGGTATAATGCTAGC 0.813 SEQ ID NO: 34 13 TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGC 1.000 SEQ ID NO: 35

[0145] The prepared plasmids were introduced into the Vibrio sp. DHG strain, and then, colonies were randomly selected, and cultured in a minimal medium. Fluorescence per cell of the strains as cultured were compared with each other. The results are shown in FIG. 7.

[0146] As shown in FIG. 7, it was identified that the fluorescence changes by about 20 times depending on the promoter sequence. Thus, it is expected that the promoter may be used to regulate the expression level of the target gene required for bio compound production.

Example 8. Quantitative Control of Translation by Changing 5' UTR Sequence

[0147] In the regulation of gene expression in the translation stage, the affinity between the mRNA to be translated and the ribosome responsible for the translation is very important. The most decisive factor to determine this affinity is the 5' UTR sequence, which determines the overall translation efficiency. According to the study of Escherichia coli, it was possible to quantitatively control an amount of a target protein by changing the 5' UTR sequence. Conversely, 5' UTR may be designed to produce the amount of the target protein. This method is provided as a web-based tool.

[0148] Likewise, in order to regulate translation in the Vibrio sp. DHG strain, the 5' UTR sequence linked to the sGFP gene was changed. In this connection, a UTR Library designer (10.1016/j.ymben.2012.10.006, 10.1038/srep04515) program was used to build an unbiased library. The constructed 5' UTR library is shown in Table 2.

TABLE-US-00002 TABLE 2 Measured flourescence Samples Sequence (5'->3') .DELTA.G.sub.UTR intensity SEQ ID NO: Template ACGGAGAWTGCTYAAKSAGTCSTTT SEQ ID NO: 36 1 ACGGAGATTGCTTAAGCAGTCGTTT 0.28 0.02 SEQ ID NO: 37 2 ACGGAGAATGCTTAATCAGTCGTTT 1.13 0.03 SEQ ID NO: 38 3 ACGGAGATTGCTTAATCAGTCCTTT 5.98 0.04 SEQ ID NO: 39 4 ACGGAGATTGCTTAAGCAGTCGTTT 0.28 0.04 SEQ ID NO: 40 5 ACGGAGAATGCTCAATGAGTCGTTT -1.22 0.12 SEQ ID NO: 41 6 ACGGAGATTGCTTAATGAGTCGTTT -2.07 0.17 SEQ ID NO: 42 7 ACGGAGAATGCTTAATGAGTCGTTT -2.17 0.18 SEQ ID NO: 43 8 ACGGAGAATGCTTAATGAGTCGTTT -2.17 0.21 SEQ ID NO: 44 9 ACGGAGATTGCTTAATGAGTCGTTT -2.07 0.24 SEQ ID NO: 45 10 ACGGAGAATGCTCAAGGAGTCGTTT -7.22 0.69 SEQ ID NO: 46 11 ACGGAGAATGCTTAAGGAGTCGTTT -8.67 0.85 SEQ ID NO: 47 12 ACGGAGATTGCTTAAGGAGTCCTTT -4.27 0.87 SEQ ID NO: 48 13 ACGGAGAATGCTTAAGGAGTCGTTT -8.67 0.98 SEQ ID NO: 49 14 ACGGAGATTGCTTAAGGAGTCGTTT -8.57 1.00 SEQ ID NO: 50

[0149] After transforming the sgfp expression plasmid designed to control 5' UTR shown in Table 2 to the Vibrio sp. DHG strain, colonies were randomly selected and cultured in minimal medium. Fluorescence per cell of the strains cultured was compared with each other. The results are shown in FIG. 8.

[0150] As shown in FIG. 8, the fluorescence increased by about 70 times based on the 5' UTR sequences. Further, from a result of identifying the 5' UTR sequence, it was identified that the fluorescence per cell had a very high correlation (R.sup.2=0.8145) with the predicted expression value based on the UTR sequence. The results indicate that translational regulation of the Vibrio sp. DHG strain may be used to optimize various compound production circuits.

Example 9. Genome Engineering of Vibrio sp. DHG Strain

[0151] 9-1. Genome Analysis of Vibrio sp. DHG Strain

[0152] In general, microorganisms do not produce only the target compound, but produce by-products (acetate, lactate, succinate, formate) for several purposes (such as achieving ATP and NAD/NADH balance). However, in order to maximize the production of the desired compound, it is necessary to prevent the production of such by-products. The most representative method for suppressing the production of by-products is to delete the gene from the genome of the microorganism.

[0153] The genome engineering of Vibrio may be achieved using single-stranded DNA oligos when expressing SXT recombinase in the genome (doi: 10.1101/130088). To this end, the presence or absence of SXT recombinase in the Vibrio sp. DHG strain was analyzed based on the decoded genome sequence.

[0154] Thus, it was confirmed that the beta and exo proteins constituting the SXT recombinase were present in the Vibrio sp. DHG strain. The beta protein is represented by the amino acid sequence of SEQ ID NO: 2, and the gene encoding the beta protein is represented by the nucleotide sequence of SEQ ID NO: 3. Further, the exo protein is represented by the amino acid sequence of SEQ ID NO: 4. The gene encoding the exo protein is represented by the nucleotide sequence of SEQ ID NO: 5.

[0155] 9-2. Plasmid Construction for Gamma Protein Expression

[0156] The SXT recombinase requires the help of gamma proteins in addition to the beta and exo proteins. As a result of analyzing the genome of the Vibrio sp. DHG strain, it was identified that there was no gamma protein in the Vibrio sp. DHG strain. Thus, a gene encoding a gamma protein derived from lambda phage was introduced into the Vibrio sp. DHG strain. The gene encoding the gamma protein is represented by the nucleotide sequence of SEQ ID NO: 6. Specifically, in order to efficiently express these genes, the recombinant enzyme was expressed in the tac promoter showing high transcription efficiency in the Vibrio sp. DHG strain. Synthetic 5' UTR was designed to have maximum translation efficiency. As a result, plasmid pACYCA_SXT was constructed. The plasmid pACYCA_SXT is represented by the cleavage map shown in FIG. 9. The sequence is represented by the nucleotide sequence of SEQ ID NO: 51.

[0157] 9-3. Construction of Plasmids Containing Antibiotic Resistance Gene and flp Flippase Gene

[0158] To selectively isolate the recombinant cells, antibiotic resistance genes were inserted thereto upon gene deletion and introduction. The antibiotic resistance gene should be easily re-deleted for further genome engineering. Gene deletion method was configured to express the flp flippase derived from Saccharomyces cerevisiae as represented by SEQ ID NO: 7 or 8 to recognize the FRT sequences on either side of the selection marker so that the deletion occurs. In Escherichia coli, generally, a plasmid called pCP20 is used. Since the plasmid is not transformed into the Vibrio sp. DHG strain, thus a new plasmid pRSF_FLP was constructed. The plasmid pRSF_FLP was designed to continuously express flp flippase for rapid FRT sequence recognition and deletion of selectable markers and therefore, was designed to be expressed under the constant promoter J23100. Further, the plasmid pRSF_FLP designed a synthetic 5' UTR with high translation efficiency to facilitate protein expression. The prepared plasmid pRSF_FLP is represented by the cleavage map shown in FIG. 10. The entire sequence is represented by the nucleotide sequence of SEQ ID NO: 52.

[0159] 9-4. Genome Engineering of Vibrio sp. DHG Strain

[0160] A schematic diagram of the genome engineering method for the Vibrio sp. DHG strain is shown in FIG. 11.

[0161] Specifically, the plasmid pACYCA_SXT prepared in Example 9-2 was transformed into the Vibrio sp. DHG strain to be genome engineered which was cultured. The cultured Vibrio sp. DHG strain was inoculated into a liquid LBv2+ampicillin (100 ug/mL) medium and then incubated overnight. Further, the strain was inoculated at a 1/100 dilution ratio into a fresh medium to which 1 mM IPTG was added for additional cultivation. When the OD of the cultured Vibrio sp. DHG strain reached 0.7 to 0.8, the medium was cooled for 10 minutes using ice or the like. We centrifuged the cooled Vibrio sp. DHG strain to obtain only cells which were washed twice with an electroporation buffer. A double stranded DNA was introduced into the washed Vibrio sp. DHG strain by electroporation (0.8 kV). In the double stranded DNA, a homology of 1 to 3 kb around the targeted gene is placed next to both sides of the selection marker (preferably the cat gene) (FIG. 12). 1 mL of BHI recovery medium was added to the transformed Vibrio sp. DHG strain which was incubated at 37.degree. C. for 3 hours therein.

[0162] The cultured strains were plated on plates containing antibiotics corresponding to selection markers and incubated for 6 hours. Colony PCR checked whether the genome engineering occurred in cells that showed antibiotic resistance. The selection marker was removed by introducing the pRSF_FLP as a plasmid for expression of the gamma protein into cells being subjected to the genome engineering. Colony PCR was finally used to identify whether the selection marker was deleted therefrom. In this connection, the cell lacking the selection marker is a Vibrio sp. DHG strain transformed with a gene encoding a gamma protein and an antibiotic resistance gene. The strain expresses the SXT recombinase, thereby allowing genome engineering using the single-stranded DNA oligos.

Example 10. Alginic Acid Metabolizing Enzyme Screening by Genomic Analysis of Vibrio sp. DHG Strain

[0163] The possibility of metabolism of the alginic acid in the sea microalgae means that the alginic acid metabolic pathway is present in the Vibrio sp. DHG strain. Alginic acid is known to be converted into pyruvate and G3P through metabolism.

[0164] In order to search for enzymes related to alginic acid metabolism, genome sequence analysis was performed on the Vibrio sp. DHG strain. It was confirmed that in the Vibrio sp. DHG strain, enzymes essential for alginic acid metabolism as follows are present.

[0165] Alginate lyase 1 to 5

[0166] DEHU reductase (2-hydroxy-3-oxopropionate reductase) 1

[0167] 2-dehydro-3-deoxygluconate kinase 1 to 2

Example 11. Preparation of Transformed Strain for Lycopene Production Using Vibrio sp. DHG Strain

[0168] As the Vibrio sp. DHG strain may metabolize alginic acid as identified in Example 10, a plasmid containing an enzyme gene of biosynthesis of lycopene is introduced into the Vibrio sp. DHG strain which may be genome engineered in Example 9-4, thereby producing a transformed strain for the lycopene production.

[0169] Specifically, the plasmid containing the lycopene biosynthetic enzyme gene used (i) a plasmid pACYC_idi_ispA_crtEBI (FIG. 17; SEQ ID NO: 53) which is improved based on the plasmid (pCDF_idi_ispA_crtEBI, doi: 10.1016/j.ymben.2016.10.003) developed to produce lycopene in E. coli, or (ii) a plasmid pACYC_idi_ispA_crtEBI_dxs (FIG. 18, SEQ ID NO: 54) which is newly constructed to further express the dxs gene derived from E. coli. The crtEBI gene (SEQ ID NO: 9) contained in the plasmid is derived from Lamprocystis purpurea. The idi (SEQ ID NO: 10), ispA (SEQ ID NO: 11) and dxs gene (SEQ ID NO: 12) are derived from E. coli. K-12 W3110. Further, the genes are designed to increase the gene expression thereof by the J23100 promoter as a constant promoter, and the synthetic 5' UTR with a high translation efficiency.

[0170] The transformed strain VDHG102 for lycopene production was prepared by transforming the Vibrio sp. DHG strain from which the dns gene was deleted using the plasmid pACYC_idi_ispA_crtEBI by the method of Example 5. After incubating the produced lycopene production strain and alginic acid for 9 hours, a lycopene production amount was measured. The composition of the medium used for the culture is as follows.

[0171] NaCl 30 g/L

[0172] (NH.sub.4)2SO.sub.4 5 g/L

[0173] K.sub.2HPO.sub.4 2 g/L

[0174] MgSO.sub.47H.sub.2O 0.5 g/L

[0175] Alginate 10 g/L

[0176] ATCC Trace mineral solution 2 ml/L

[0177] Chloramphenicol 10 ug/mL

[0178] Experimental group VDHG103 was achieved by transforming the Vibrio sp. DHG strain from which the dns gene is deleted using the plasmid pACYC_idi_ispA_crtEBI_dxs and corresponds to the lycopene production strain in which the dxs gene derived from E. coli is further expressed. Experimental group VDHG103 (Alg 20) was achieved by incubating the VDHG103 strain while the alginic acid was additionally supplied to the medium during the culture. Lycopene production by the transformed strain for lycopene production is shown in FIG. 13.

[0179] As shown in FIG. 13, it is identified that the experimental group VDHG102 has 3.74 mg/L of lycopene production. Experimental group VDHG103 additionally expressing the dxs gene derived from E. coli has 6 mg/L of lycopene production. The experimental group VDHG103 (Alg 20) in which an additional supply of the alginic acid occurred has about 9.4 mg/L of lycopene production. The results suggest that the Vibrio sp. DHG strain can be used universally in the production of high value-added compounds such as lycopene.

Example 12. Preparation of Transformed Strain for Producing 2,3-Butanediol Using Vibrio sp. DHG Strain

[0180] 2,3-butanediol is an industrially effective compound used in plastic synthesis, antifreeze, and pesticides. 2,3-butanediol is subjected to chemical conversion for being widely used for fuel additives and rubber synthesis. It is known that 2,3-butanediol may be produced by introducing and expressing operon (budACB) composed of budA, budB and budC derived from Klebsiella pneumoniae.

[0181] Thus, the budACB operon composed of budA (SEQ ID NO: 13), budB (SEQ ID NO: 14), and budC gene (SEQ ID NO: 15) was introduced into the Vibrio sp. DHG strain as prepared in Example 9-2. Thus, a 2,3-butanediol production strain was produced. Specifically, a tac promoter and the 5' UTR with a high translation efficiency were designed for efficient expression of the budACB operon. The plasmid pACYC_BudACB was designed using the tac promoter and the 5' UTR. The plasmid pACYC_BudACB is represented by the cleavage map shown in FIG. 14. The entire sequence is represented by the nucleotide sequence of SEQ ID NO: 55. The plasmid pACYC_BudACB was introduced into the Vibrio sp. DHG strain prepared in Example 9-2 to transform the latter to prepare a 2,3-butanediol production strain (Experimental Group 1).

[0182] Also, in order to increase the productivity and yield of the 2,3-butanediol, the genome engineering method of Example 9 may be used to sequentially delete the metabolite producing genes ldhA (lactic acid), frdABCD operon (succinate production) and pflB (conversion of pyruvate to Acetyl-CoA) from the Vibrio sp. DHG strain having the budACB operon introduced thereto, by using the plasmid comprising the flp flippase gene of Example 9-2 having a competing relationship with 2,3-butanediol production. Thus, the 2,3-butanediol production strain with increased production efficiency was prepared (Experimental group 2). The IdhA gene is represented by the nucleotide sequence of SEQ ID NO: 16. The genes constituting the frdABCD operon may be represented by nucleotide sequences of SEQ ID NOs: 17 to 20 respectively. The pflB gene is represented by the nucleotide sequence of SEQ ID NO: 21. Colony PCR was performed to identify the gene deletion result in the 2,3-butanediol production strain with increased production efficiency. The results are shown in FIG. 15.

[0183] As shown in FIG. 15, in the 2,3-butanediol production transformed strain with increased production efficiency, the metabolite producing genes ldhA, frdABCD operon and pflB having a competing relationship with 2,3-butanediol production are deleted.

[0184] Strains of Experimental Groups 1 and 2 were incubated at 30.degree. C. and 250 rpm using media having following compositions, respectively. After the end of the culture, metabolites and the production of 2,3-butanediol were measured. The results are shown in FIG. 16. The control is a Vibrio sp. DHG strain prepared in Example 9-2 and is free of the plasmid pACYC_BudACB.

[0185] The composition of the medium is as follows. Compositions of alginic acid, mannitol, and glucose as obtained from the brown microalgae were used. The alginic acid, mannitol, and glucose were mixed with each other to obtain the carbon source. The total amount thereof was set to 50 g/L.

[0186] NaCl 10 g/L

[0187] (NH.sub.4)2SO.sub.4 5 g/L

[0188] Potassium buffer 100 mM (pH 7)

[0189] Yeast extracts 5 g/L

[0190] MgSO.sub.4.7H.sub.2O 0.5 g/L

[0191] Carbon source 50 g/L

[0192] DSMZ Trace element solution 2 ml/L

[0193] As shown in FIG. 16, it is identified that the control without the introduction of the plasmid pACYC_BudACB has no 2,3-butanediol production. On the other hand, Experimental Group 1 as a strain to which the plasmid pACYC_BudACB was introduced, produced 2,3-butanediol. Experimental group 2 as a strain from which the enzymes having competitive metabolic pathways are deleted shows the production of 2,3-butanediol as significantly increased. The results suggest that the Vibrio sp. DHG strain may be used universally in the production of high value-added compounds such as 2,3-butanediol.

[0194] Overall, the present inventors have isolated the Vibrio sp. DHG strains from seawater. The strain grows much faster in the minimal medium and nutrient-rich medium than microorganisms such as Escherichia coli. The strain is resistant to high initial sugar/salt concentrations. Further, the strain may be transformed using a conventional plasmid system for improving E. coli. Thus, the genome engineering of the Vibrio sp. DHG strain results in producing the lycopene and 2,3-butanediol. The Vibrio sp. DHG strain according to the present disclosure may be used in various production fields high value-added compounds using microorganisms.

[0195] From the foregoing, it will be appreciated that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications may be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

[0196] [Accession Number]

[0197] Depositary: Korea Research Institute of Bioscience and Biotechnology

[0198] Accession number: KCTC13239BP

[0199] Deposit Date: 2017 Apr. 6

Sequence CWU 1

1

6911535DNAUnknownVibrio sp. dhg 1catggctcag attgaacgct ggcggcaggc ctaacacatg caagtcgagc ggaaacgagt 60taactgaacc ttcgggggac gttaacggcg tcgagcggcg gacgggtgag taatgcctag 120gaaattgccc tgatgtgggg gataaccatt ggaaacgatg gctaataccg catgatgcct 180acgggccaaa gagggggacc ttcgggcctc tcgcgtcagg atatgcctag gtgggattag 240ctagttggtg aggtaagggc tcaccaaggc gacgatccct agctggtctg agaggatgat 300cagccacact ggaactgaga cacggtccag actcctacgg gaggcagcag tggggaatat 360tgcacaatgg gcgcaagcct gatgcagcca taccgcgtgt gtgaagaagg ccttcgggtt 420gtaaagcact ttcagtcgtg aggaaggtag tgtgtttaat agatgcatta tttgacgtta 480gcgacagaag aagcaccggc taactccgtg ccagcagccg cggtaatacg gagggtgcga 540gcgttaatcg gaattactgg gcgtaaagcg catgcaggtg gtttgttaag tcagatgtga 600aagcccgggg ctcaacctcg gaatagcatt tgaaactggc agactagagt actgtagagg 660ggggtagaat ttcaggtgta gcggtgaaat gcgtagagat ctgaaggaat accggtggcg 720aaggcggccc cctggacaga tactgacact cagatgcgaa agcgtgggga gcaaacagga 780ttagataccc tggtagtcca cgccgtaaac gatgtctact tggaggttgt ggccttgagc 840cgtggctttc ggagctaacg cgttaagtag accgcctggg gagtacggtc gcaagattaa 900aactcaaatg aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgatgc 960aacgcgaaga accttaccta ctcttgacat ccagagaact ttccagagat ggattggtgc 1020cttcgggaac tctgagacag gtgctgcatg gctgtcgtca gctcgtgttg tgaaatgttg 1080ggttaagtcc cgcaacgagc gcaaccctta tccttgtttg ccagcgagta atgtcgggaa 1140ctccagggag actgccggtg ataaaccgga ggaaggtggg gacgacgtca agtcatcatg 1200gcccttacga gtagggctac acacgtgcta caatggcgca tacagagggc ggccaacttg 1260cgaaagtgag cgaatcccaa aaagtgcgtc gtagtccgga ttggagtctg caactcgact 1320ccatgaagtc ggaatcgcta gtaatcgtgg atcagaatgc cacggtgaat acgttcccgg 1380gccttgtaca caccgcccgt cacaccatgg gagtgggctg caaaagaagt aggtagttta 1440accttcgggg gacgcttacc actttgtggt tcatgactgg ggtgaagtcg taacaaggta 1500gcgctagggg aacctggcgc tggatcacct cctta 15352272PRTUnknownVibrio sp. dhg 2Met Glu Lys Pro Lys Leu Ile Gln Arg Phe Ala Glu Arg Phe Ser Val1 5 10 15Asp Pro Asn Lys Leu Phe Asp Thr Leu Lys Ala Thr Ala Phe Lys Gln 20 25 30Arg Asp Gly Ser Ala Pro Thr Asn Glu Gln Met Met Ala Leu Leu Val 35 40 45Val Ala Asp Gln Tyr Gly Leu Asn Pro Phe Thr Lys Glu Ile Phe Ala 50 55 60Phe Pro Asp Lys Gln Ala Gly Ile Ile Pro Val Val Gly Val Asp Gly65 70 75 80Trp Ser Arg Ile Ile Asn Gln His Asp Gln Phe Asp Gly Met Glu Phe 85 90 95Lys Thr Ser Glu Asn Lys Val Ser Leu Asp Gly Ala Lys Glu Cys Pro 100 105 110Glu Trp Met Glu Cys Ile Ile Tyr Arg Arg Asp Arg Ser His Pro Val 115 120 125Lys Ile Thr Glu Tyr Leu Asp Glu Val Tyr Arg Pro Pro Phe Glu Gly 130 135 140Asn Gly Lys Asn Gly Pro Tyr Arg Val Asp Gly Pro Trp Gln Thr His145 150 155 160Thr Lys Arg Met Leu Arg His Lys Ser Met Ile Gln Cys Ser Arg Ile 165 170 175Ala Phe Gly Phe Val Gly Ile Phe Asp Gln Asp Glu Ala Glu Arg Ile 180 185 190Ile Glu Gly Gln Ala Thr His Val Val Glu Pro Ser Val Ile Pro Pro 195 200 205Glu Gln Val Asp Asp Arg Thr Arg Gly Leu Val Tyr Lys Leu Ile Glu 210 215 220Arg Ala Glu Ala Ser Asn Ala Trp Asn Ser Ala Leu Glu Tyr Ala Asn225 230 235 240Glu His Phe Gln Gly Val Glu Leu Thr Phe Ala Lys Gln Glu Ile Ile 245 250 255Asn Ala Gln Gln Gln Ala Ala Lys Ala Leu Thr Gln Pro Leu Ala Ser 260 265 2703819DNAUnknownVibrio sp. dhg 3atggaaaaac caaagctaat tcaacgcttt gctgagcgct ttagtgtcga tccaaacaag 60ttgttcgata ccctaaaagc aacagcattc aagcaacgtg acggtagtgc accgaccaat 120gagcagatga tggcgctctt ggtggttgca gatcagtacg gcttgaaccc tttcaccaaa 180gagatttttg cgttccctga taaacaagct gggattattc cagtggtagg tgtcgatgga 240tggtctcgca tcattaatca acacgaccag tttgatggca tggagtttaa gacttcagaa 300aacaaagtct ccctggatgg cgcgaaagaa tgcccggaat ggatggaatg catcatctat 360cggcgcgacc gttcgcaccc agtcaaaatc actgaatacc tggatgaagt ctatcgaccg 420ccttttgaag gtaacggcaa aaatggccct taccgggtag atggtccatg gcagacgcac 480actaagcgaa tgctaagaca taaatccatg atccagtgtt cccgcattgc gtttggcttt 540gtgggaattt tcgatcaaga cgaagcggag cgaattatcg aaggccaagc aacacacgtt 600gttgagccat cggtgattcc acccgagcaa gttgatgatc gaacccgagg gcttgtttac 660aagcttatcg agcgggcgga agcttcaaac gcttggaata gtgcattgga atatgccaat 720gaacattttc aaggtgttga actgacgttt gcgaaacaag aaataattaa tgcacagcaa 780caagcagcca aagcgctcac acagccttta gcttcttag 8194338PRTUnknownVibrio sp. dhg 4Met Lys Val Ile Asp Leu Ser Gln Arg Thr Pro Ala Trp His Gln Trp1 5 10 15Arg Ile Ala Gly Val Thr Ala Ser Glu Ala Pro Ile Ile Met Gly Arg 20 25 30Ser Pro Tyr Lys Thr Pro Trp Arg Leu Trp Ala Glu Lys Thr Gly Phe 35 40 45Val Leu Pro Glu Asp Leu Ser Asn Asn Pro Asn Val Leu Arg Gly Ile 50 55 60Arg Leu Glu Pro Gln Ala Arg Arg Ala Phe Glu Asn Ala His Asn Asp65 70 75 80Phe Leu Leu Pro Leu Cys Ala Glu Ala Asp His Asn Ala Ile Phe Arg 85 90 95Ala Ser Phe Asp Gly Ile Asn Asp Ala Gly Glu Pro Val Glu Leu Lys 100 105 110Cys Pro Cys Gln Ser Val Phe Glu Asp Val Gln Ala His Arg Glu Gln 115 120 125Ser Glu Ala Tyr Gln Leu Tyr Trp Val Gln Val Gln His Gln Ile Leu 130 135 140Val Ala Asn Ser Thr Arg Gly Trp Leu Val Phe Tyr Phe Glu Asp Gln145 150 155 160Leu Ile Glu Phe Glu Ile Gln Arg Asp Ala Ala Phe Leu Thr Glu Leu 165 170 175Gln Glu Thr Ala Leu Gln Phe Trp Glu Leu Val Gln Thr Lys Lys Glu 180 185 190Pro Ser Lys Cys Pro Glu Gln Asp Cys Phe Val Pro Lys Gly Glu Ala 195 200 205Gln Tyr Arg Trp Thr Ser Leu Ser Arg Gln Tyr Cys Ser Ala His Ala 210 215 220Glu Val Val Arg Leu Glu Asn His Ile Lys Ser Leu Lys Glu Glu Met225 230 235 240Arg Glu Ala Gln Ser Lys Leu Val Ala Met Met Gly Asn Tyr Ala His 245 250 255Ala Asp Tyr Ala Gly Val Lys Leu Ser Arg Tyr Met Met Ala Gly Thr 260 265 270Val Asp Tyr Lys Gln Leu Ala Thr Asp Lys Leu Gly Glu Leu Asp Glu 275 280 285Gln Val Leu Ala Ala Tyr Arg Lys Ala Pro Gln Glu Arg Leu Arg Ile 290 295 300Ser Thr Asn Lys Pro Glu Gln Pro Val Glu Thr Pro Ile Lys Ile Ser305 310 315 320Leu Glu Gln Glu Asn Leu Val Leu Pro Gly Asp Ser Pro Ser Ser Phe 325 330 335Tyr Phe51017DNAUnknownVibrio sp. dhg 5atgaaggtta tcgacctatc acaacgtact cctgcatggc accagtggcg cattgcaggg 60gttacggcat ctgaagcccc aattattatg gggcgttcac cctacaaaac accttggcga 120ttatgggcag aaaaaaccgg attcgtatta ccggaagacc tatcgaataa tccaaatgtg 180cttcgaggta tacggttgga gcctcaagca aggcgagcat ttgagaatgc gcataatgac 240tttcttctgc cgttatgtgc agaagccgat cataacgcaa tctttcgagc cagctttgat 300ggcatcaacg atgcgggcga acctgttgaa ctgaaatgtc cttgccagtc agtttttgag 360gatgtgcaag ctcaccgaga acaaagtgag gcgtaccagt tgtattgggt gcaagtacag 420caccaaatac tggtcgccaa tagcacgcgt ggttggttgg tattctattt tgaggatcaa 480ctgattgagt ttgaaataca acgagacgcg gcgttcttaa ctgagttgca agaaacagcg 540cttcagtttt gggagttagt acagaccaaa aaagaaccgt caaaatgccc tgagcaagat 600tgttttgttc ccaagggtga agcccaatac cgttggacat cgctatcacg gcagtattgc 660tcagcacatg ccgaagtggt ccgactggaa aaccacatta aatctttgaa agaggaaatg 720cgagaagctc agtcgaaatt ggtcgctatg atgggtaact acgctcatgc cgactatgct 780ggggtcaaac tcagccgcta catgatggca ggtacggtgg actataagca attggccacc 840gataagttag gcgagctgga tgaacaggtt ttagctgctt accgaaaagc gccacaagag 900cggttgcgca ttagcaccaa taagccagag cagcccgttg aaacaccaat caaaatcagc 960cttgagcaag agaacttggt tctgccaggt gactcgccga gctcatttta cttttaa 10176417DNAUnknownLamda phage 6atggatatta atactgaaac tgagatcaag caaaagcatt cactaacccc ctttcctgtt 60ttcctaatca gcccggcatt tcgcgggcga tattttcaca gctatttcag gagttcagcc 120atgaacgctt attacattca ggatcgtctt gaggctcaga gctgggcgcg tcactaccag 180cagctcgccc gtgaagagaa agaggcagaa ctggcagacg acatggaaaa aggcctgccc 240cagcacctgt ttgaatcgct atgcatcgat catttgcaac gccacggggc cagcaaaaaa 300tccattaccc gtgcgtttga tgacgatgtt gagtttcagg agcgcatggc agaacacatc 360cggtacatgg ttgaaaccat tgctcaccac caggttgata ttgattcaga ggtataa 41771272DNASaccharomyces cerevisiae 7atgccacaat ttggtatatt atgtaaaaca ccacctaagg tgcttgttcg tcagtttgtg 60gaaaggtttg aaagaccttc aggtgagaaa atagcattat gtgctgctga actaacctat 120ttatgttgga tgattacaca taacggaaca gcaatcaaga gagccacatt catgagctat 180aatactatca taagcaattc gctgagtttc gatattgtca ataaatcact ccagtttaaa 240tacaagacgc aaaaagcaac aattctggaa gcctcattaa agaaattgat tcctgcttgg 300gaatttacaa ttattcctta ctatggacaa aaacatcaat ctgatatcac tgatattgta 360agtagtttgc aattacagtt cgaatcatcg gaagaagcag ataagggaaa tagccacagt 420aaaaaaatgc ttaaagcact tctaagtgag ggtgaaagca tctgggagat cactgagaaa 480atactaaatt cgtttgagta tacttcgaga tttacaaaaa caaaaacttt ataccaattc 540ctcttcctag ctactttcat caattgtgga agattcagcg atattaagaa cgttgatccg 600aaatcattta aattagtcca aaataagtat ctgggagtaa taatccagtg tttagtgaca 660gagacaaaga caagcgttag taggcacata tacttcttta gcgcaagggg taggatcgat 720ccacttgtat atttggatga atttttgagg aattctgaac cagtcctaaa acgagtaaat 780aggaccggca attcttcaag caataaacag gaataccaat tattaaaaga taacttagtc 840agatcgtaca ataaagcttt gaagaaaaat gcgccttatt caatctttgc tataaaaaat 900ggcccaaaat ctcacattgg aagacatttg atgacctcat ttctttcaat gaagggccta 960acggagttga ctaatgttgt gggaaattgg agcgataagc gtgcttctgc cgtggccagg 1020acaacgtata ctcatcagat aacagcaata cctgatcact acttcgcact agtttctcgg 1080tactatgcat atgatccaat atcaaaggaa atgatagcat tgaaggatga gactaatcca 1140attgaggagt ggcagcatat agaacagcta aagggtagtg ctgaaggaag catacgatac 1200cccgcatgga atgggataat atcacaggag gtactagact acctttcatc ctacataaat 1260agacgcatat aa 12728423PRTSaccharomyces cerevisiae 8Met Pro Gln Phe Gly Ile Leu Cys Lys Thr Pro Pro Lys Val Leu Val1 5 10 15Arg Gln Phe Val Glu Arg Phe Glu Arg Pro Ser Gly Glu Lys Ile Ala 20 25 30Leu Cys Ala Ala Glu Leu Thr Tyr Leu Cys Trp Met Ile Thr His Asn 35 40 45Gly Thr Ala Ile Lys Arg Ala Thr Phe Met Ser Tyr Asn Thr Ile Ile 50 55 60Ser Asn Ser Leu Ser Phe Asp Ile Val Asn Lys Ser Leu Gln Phe Lys65 70 75 80Tyr Lys Thr Gln Lys Ala Thr Ile Leu Glu Ala Ser Leu Lys Lys Leu 85 90 95Ile Pro Ala Trp Glu Phe Thr Ile Ile Pro Tyr Tyr Gly Gln Lys His 100 105 110Gln Ser Asp Ile Thr Asp Ile Val Ser Ser Leu Gln Leu Gln Phe Glu 115 120 125Ser Ser Glu Glu Ala Asp Lys Gly Asn Ser His Ser Lys Lys Met Leu 130 135 140Lys Ala Leu Leu Ser Glu Gly Glu Ser Ile Trp Glu Ile Thr Glu Lys145 150 155 160Ile Leu Asn Ser Phe Glu Tyr Thr Ser Arg Phe Thr Lys Thr Lys Thr 165 170 175Leu Tyr Gln Phe Leu Phe Leu Ala Thr Phe Ile Asn Cys Gly Arg Phe 180 185 190Ser Asp Ile Lys Asn Val Asp Pro Lys Ser Phe Lys Leu Val Gln Asn 195 200 205Lys Tyr Leu Gly Val Ile Ile Gln Cys Leu Val Thr Glu Thr Lys Thr 210 215 220Ser Val Ser Arg His Ile Tyr Phe Phe Ser Ala Arg Gly Arg Ile Asp225 230 235 240Pro Leu Val Tyr Leu Asp Glu Phe Leu Arg Asn Ser Glu Pro Val Leu 245 250 255Lys Arg Val Asn Arg Thr Gly Asn Ser Ser Ser Asn Lys Gln Glu Tyr 260 265 270Gln Leu Leu Lys Asp Asn Leu Val Arg Ser Tyr Asn Lys Ala Leu Lys 275 280 285Lys Asn Ala Pro Tyr Ser Ile Phe Ala Ile Lys Asn Gly Pro Lys Ser 290 295 300His Ile Gly Arg His Leu Met Thr Ser Phe Leu Ser Met Lys Gly Leu305 310 315 320Thr Glu Leu Thr Asn Val Val Gly Asn Trp Ser Asp Lys Arg Ala Ser 325 330 335Ala Val Ala Arg Thr Thr Tyr Thr His Gln Ile Thr Ala Ile Pro Asp 340 345 350His Tyr Phe Ala Leu Val Ser Arg Tyr Tyr Ala Tyr Asp Pro Ile Ser 355 360 365Lys Glu Met Ile Ala Leu Lys Asp Glu Thr Asn Pro Ile Glu Glu Trp 370 375 380Gln His Ile Glu Gln Leu Lys Gly Ser Ala Glu Gly Ser Ile Arg Tyr385 390 395 400Pro Ala Trp Asn Gly Ile Ile Ser Gln Glu Val Leu Asp Tyr Leu Ser 405 410 415Ser Tyr Ile Asn Arg Arg Ile 42093333DNAUnknownLamprocystis purpurea 9atggtatctg gctcaaaggc tggcgtctcg ccacatcgcg aaattgaagt gatgcgccag 60agcattgatg atcatctggc gggcctgctg ccggaaaccg atagccagga tattgtgagc 120ctggcgatgc gcgaaggcgt gatggcgccg ggcaaacgca ttcgcccgct gctgatgctg 180ctggcggcgc gcgatctgcg ctatcagggc agtatgccga ccctgctgga tctggcgtgc 240gcggtggaac tgacccatac cgcgagcctg atgctggatg atatgccgtg catggataac 300gcggaactgc gccgcggcca gccgaccacc cataaaaaat ttggcgaaag cgtggcgatt 360ctggcgagcg tgggcctgct gagcaaagcg tttggcctga ttgcggcgac cggcgatctg 420ccgggcgaac gccgcgcgca ggcggtgaac gaactgagca ccgcggtggg cgtgcagggc 480ctggtgctgg gccagtttcg cgatctgaac gatgcggcgc tggatcgcac cccggatgcg 540attctgagca ccaaccatct gaaaaccggc attctgttta gcgcgatgct gcagattgtg 600gcgattgcga gcgcgagcag cccgagcacc cgcgaaaccc tgcacgcgtt tgcgctggat 660tttggccagg cgtttcagct gctggatgat ctgcgcgatg atcatccgga aaccggcaaa 720gatcgcaaca aagatgcggg caaaagcacc ctggtgaacc gcctgggcgc ggatgcggcg 780cgccagaaac tgcgcgaaca tattgatagc gcggataaac atctgacctt tgcgtgcccg 840cagggcggcg cgattcgcca gtttatgcat ctgtggtttg gccatcatct ggcggattgg 900agcccggtga tgaaaattgc gtaaatgtcc caacccccct tgctagacca cgcaacccag 960acgatggcga acggcagcaa gagctttgcg accgcggcga aactgtttga tccggcgacc 1020cgccgcagcg tgctgatgct gtatacctgg tgccgccatt gcgatgatgt gattgatgat 1080cagacgcatg gctttgcgag cgaagcggcg gcggaagaag aagcgaccca gcgcctggcg 1140cgcctgcgca ccctgaccct ggcggcgttt gaaggcgcgg aaatgcagga cccggcgttt 1200gcggcgtttc aggaagtggc gctgacccac ggcattaccc cgcgcatggc gctggatcat 1260ctggatggct ttgcgatgga tgtggcgcag acccgctatg tgacctttga agataccctg 1320cgctattgct atcatgtggc gggcgtggtg ggcctgatga tggcgcgcgt gatgggcgtg 1380cgcgatgaac gcgtgctgga tcgcgcgtgc gatctgggcc tggcgtttca gctgaccaac 1440attgcgcgcg atattattga tgatgcggcg attgatcgct gctatctgcc ggcggaatgg 1500ctgcaggatg cgggcctgac cccggaaaac tatgcggcgc gcgaaaaccg cgcggcgctg 1560gcgcgcgtgg cggaacgcct gattgatgcg gcggaaccgt attatattag cagccaggcg 1620ggcctgcatg atctgccgcc gcgctgcgcg tgggcgattg cgaccgcgcg cagcgtgtat 1680cgcgaaattg gcattaaagt gaaagcggcg ggcggcagcg cgtgggatcg ccgccagcat 1740accagcaaag gcgaaaaaat tgcgatgctg atggcggcgc cgggccaggt gattcgcgcg 1800aaaaccaccc gcgtgacccc gcgcccggcg ggcctgtggc agcgcccggt gtaaatgaaa 1860aaaacggttg tgatcggcgc tgggttcggc ggcctggcgc tggcgattcg cctgcaggcg 1920gcgggcattc cgaccgtgct gctggaacag cgcgataaac cgggcggccg cgcgtatgtg 1980tggcatgatc agggctttac ctttgatgcg ggcccgaccg tgattaccga tccgaccgcg 2040ctggaagcgc tgtttaccct ggcgggccgc cgcatggaag attatgtgcg cctgctgccg 2100gtgaaaccgt tttatcgcct gtgctgggaa agcggcaaaa ccctggatta tgcgaacgat 2160agcgcggaac tggaagcgca gattacccag tttaacccgc gcgatgtgga aggctatcgc 2220cgctttctgg cgtatagcca ggcggtgttt caggaaggct atctgcgcct gggcagcgtg 2280ccgtttctga gctttcgcga tatgctgcgc gcgggcccgc agctgctgaa actgcaggcg 2340tggcagagcg tgtatcagag cgtgagccgc tttattgaag atgaacatct gcgccaggcg 2400tttagctttc atagcctgct ggtgggcggc aacccgttta ccaccagcag catttatacc 2460ctgattcatg cgctggaacg cgaatggggc gtgtggtttc cggaaggcgg caccggcgcg 2520ctggtgaacg gcatggtgaa actgtttacc gatctgggcg gcgaaattga actgaacgcg 2580cgcgtggaag aactggtggt ggcggataac cgcgtgagcc aggtgcgcct ggcggatggc 2640cgcatttttg ataccgatgc ggtggcgagc aacgcggatg tggtgaacac ctataaaaaa 2700ctgctgggcc atcatccggt gggccagaaa cgcgcggcgg cgctggaacg caaaagcatg 2760agcaacagcc tgtttgtgct gtattttggc ctgaaccagc cgcatagcca gctggcgcat 2820cataccattt gctttggccc gcgctatcgc gaactgattg atgaaatttt taccggcagc 2880gcgctggcgg atgattttag cctgtatctg catagcccgt gcgtgaccga tccgagcctg 2940gcgccgccgg gctgcgcgag cttttatgtg ctggcgccgg tgccgcatct gggcaacgcg 3000ccgctggatt gggcgcagga aggcccgaaa ctgcgcgatc gcatttttga ttatctggaa 3060gaacgctata tgccgggcct gcgcagccag ctggtgaccc agcgcatttt taccccggcg 3120gattttcatg ataccctgga tgcgcatctg ggcagcgcgt ttagcattga accgctgctg 3180acccagagcg cgtggtttcg cccgcataac cgcgatagcg atattgcgaa cctgtatctg 3240gtgggcgcgg gcacccatcc gggcgcgggc attccgggcg tggtggcgag cgcgaaagcg 3300accgcgagcc tgatgatcga agacctgcag taa

333310549DNAEscherichia coli 10atgcagaccg aacatgtgat tctgctgaac gcgcagggcg tgccgaccgg caccctggaa 60aaatatgcgg cgcataccgc ggatacccgc ctgcatctgg cgtttagcag ctggctgttt 120aacgcgaaag gccagctgct ggtgacccgc cgcgcgctga gcaaaaaagc gtggccgggc 180gtgtggacca acagcgtgtg cggccatccg cagctgggcg aaagcaacga agatgcggtg 240attcgccgct gccgctatga actgggcgtg gaaattaccc cgccggaaag catttatccg 300gattttcgct atcgcgcgac cgatccgagc ggcattgtgg aaaacgaagt gtgcccggtg 360tttgcggcgc gcaccaccag cgcgctgcag attaacgatg atgaagtgat ggattatcag 420tggtgcgatc tggcggatgt gctgcatggc attgatgcga ccccgtgggc gtttagcccg 480tggatggtga tgcaggcgac caaccgcgaa gcgcgcaaac gcctgagcgc gtttacccag 540ctgaaataa 54911900DNAEscherichia coli 11atggatttcc cgcagcagct ggaagcgtgt gtgaaacagg cgaaccaggc gctgagccgc 60tttattgcgc cgctgccgtt tcagaacacc ccggtggtgg aaaccatgca gtatggcgcg 120ctgctgggcg gcaaacgcct gcgcccgttt ctggtgtatg cgaccggcca catgtttggc 180gtgagcacca acaccctgga tgcgccggcg gcggcggtgg aatgcattca tgcgtatagc 240ctgattcatg atgatctgcc ggcgatggat gatgatgatc tgcgccgcgg cctgccgacc 300tgccatgtga aatttggcga agcgaacgcg attctggcgg gcgatgcgct gcagaccctg 360gcgtttagca ttctgagcga tgcggatatg ccggaagtga gcgatcgcga tcgcattagc 420atgattagcg aactggcgag cgcgagcggc attgcgggca tgtgcggcgg ccaggcgctg 480gatctggatg cggaaggcaa acatgtgccg ctggatgcgc tggaacgcat tcatcgccat 540aaaaccggcg cgctgattcg cgcggcggtg cgcctgggcg cgctgagcgc gggcgataaa 600ggccgccgcg cgctgccggt gctggataaa tatgcggaaa gcattggcct ggcgtttcag 660gtgcaggatg atattctgga tgtggtgggc gataccgcga ccctgggcaa acgccagggc 720gcggatcagc agctgggcaa aagcacctat ccggcgctgc tgggcctgga acaggcgcgc 780aaaaaagcgc gcgatctgat tgatgatgcg cgccagagcc tgaaacagct ggcggaacag 840agcctggata ccagcgcgct ggaagcgctg gcggattata ttattcagcg caacaaataa 900121863DNAEscherichia coli 12atgagttttg atattgccaa atacccgacc ctggcactgg tcgactccac ccaggagtta 60cgactgttgc cgaaagagag tttaccgaaa ctctgcgacg aactgcgccg ctatttactc 120gacagcgtga gccgttccag cgggcacttc gcctccgggc tgggcacggt cgaactgacc 180gtggcgctgc actatgtcta caacaccccg tttgaccaat tgatttggga tgtggggcat 240caggcttatc cgcataaaat tttgaccgga cgccgcgaca aaatcggcac catccgtcag 300aaaggcggtc tgcacccgtt cccgtggcgc ggcgaaagcg aatatgacgt attaagcgtc 360gggcattcat caacctccat cagtgccgga attggtattg cggttgctgc cgaaaaagaa 420ggcaaaaatc gccgcaccgt ctgtgtcatt ggcgatggcg cgattaccgc aggcatggcg 480tttgaagcga tgaatcacgc gggcgatatc cgtcctgata tgctggtgat tctcaacgac 540aatgaaatgt cgatttccga aaatgtcggc gcgctcaaca accatctggc acagctgctt 600tccggtaagc tttactcttc actgcgcgaa ggcgggaaaa aagttttctc tggcgtgccg 660ccaattaaag agctgctcaa acgcaccgaa gaacatatta aaggcatggt agtgcctggc 720acgttgtttg aagagctggg ctttaactac atcggcccgg tggacggtca cgatgtgctg 780gggcttatca ccacgctaaa gaacatgcgc gacctgaaag gcccgcagtt cctgcatatc 840atgaccaaaa aaggtcgtgg ttatgaaccg gcagaaaaag acccgatcac tttccacgcc 900gtgcctaaat ttgatccctc cagcggttgt ttgccgaaaa gtagcggcgg tttgccgagc 960tattcaaaaa tctttggcga ctggttgtgc gaaacggcag cgaaagacaa caagctgatg 1020gcgattactc cggcgatgcg tgaaggttcc ggcatggtcg agttttcacg taaattcccg 1080gatcgctact tcgacgtggc aattgccgag caacacgcgg tgacctttgc tgcgggtctg 1140gcgattggtg ggtacaaacc cattgtcgcg atttactcca ctttcctgca acgcgcctat 1200gatcaggtgc tgcatgacgt ggcgattcaa aagcttccgg tcctgttcgc catcgaccgc 1260gcgggcattg ttggtgctga cggtcaaacc catcagggtg cttttgatct ctcttacctg 1320cgctgcatac cggaaatggt cattatgacc ccgagcgatg aaaacgaatg tcgccagatg 1380ctctataccg gctatcacta taacgatggc ccgtcagcgg tgcgctaccc gcgtggcaac 1440gcggtcggcg tggaactgac gccgctggaa aaactaccaa ttggcaaagg cattgtgaag 1500cgtcgtggcg agaaactggc gatccttaac tttggtacgc tgatgccaga agcggcgaaa 1560gtcgccgaat cgctgaacgc cacgctggtc gatatgcgtt ttgtgaaacc gcttgatgaa 1620gcgttaattc tggaaatggc cgccagccat gaagcgctgg tcaccgtaga agaaaacgcc 1680attatgggcg gcgcaggcag cggcgtgaac gaagtgctga tggcccatcg taaaccagta 1740cccgtgctga acattggcct gccggacttc tttattccgc aaggaactca ggaagaaatg 1800cgcgccgaac tcggcctcga tgccgctggt atggaagcca aaatcaaggc ctggctggca 1860taa 186313780DNAEnterobacter aerogenes 13atgaatcatg cttcagattg cacctgtgaa gagagtctgt gtgaaacgct acgcgcgttt 60tccgctcagc atcccgatag cgtgctgtat caaacttcgc tgatgagcgc cctgctcagc 120ggcgtctacg aaggtaccac caccattgcg gacctgctga agcacggtga tttcgggctc 180ggcactttta atgaactcga cggcgagctg atcgcgttta gcagccaggt ttatcaactg 240cgtgccgacg gcagcgcgcg taaagcgcgt ccggaacaga aaacgccgtt tgcggtgatg 300acctggtttc agccgcagta ccgtaaaacc tttgaccatc cggtcagccg ccagcagctg 360catgaggtta ttgaccagca aattccttcc gacaatctgt tctgcgcgct gcgaatcgat 420ggtcatttcc gccacgccca tacccgcacc gtgcctcgtc agacgccgcc ctaccgggcg 480atgaccgacg tgctcgacga tcagccggtt ttccgcttta accagcgtga cggcgtactg 540gtcggttttc gtaccccgca gcatatgcag ggaattaacg tcgccggcta tcacgaacac 600ttcattaccg atgaccgcca gggcggcggc cacctgctgg actaccagct cgaccatggg 660gtattgacct tcggcgaaat tcataagctg atgatcgacc ttcccgccga cagcgcgttc 720ctgcaggcca atttgcatcc cgataatctc gatgccgcca tccgttcagt agaaagttag 78014771DNAEnterobacter aerogenes 14atgaaaaaag tcgcacttgt caccggcgcc ggtcagggca ttggtaaagc tatcgcgtta 60cgcctcgtga aggacggttt tgccgtggcg atcgccgatt acaatgacgt cacagcgaaa 120gccgtggcgg atgaaatcaa ccagcacggc ggccgggcaa tcgcggtcaa agtcgatgtt 180tccgaccgtg agcaggtgtt tgccgccgtc gaacaggcgc gaaaaacgct gggcggattc 240aacgtcatcg tcaataacgc cggggtcgcg ccatcaacgc ctatcgaatc cattacgccg 300gagattgtcg acaaggtcta caacatcaac gttaaagggg tgatctgggg gattcaggcg 360gcagtcgagg cctttaaaaa agaggggcac ggcggcaaaa tcatcaacgc ctgttcgcag 420gccggacacg tcggcaaccc ggaactggcg gtctacagct cgagcaaatt cgccgtacgc 480ggtttaacgc aaaccgccgc tcgcgacctg gcgccgctgg gtattaccgt taacggctac 540tgcccgggga ttgtgaaaac gccgatgtgg gccgagatcg atcgtcaggt atccgaagcg 600gcgggtaaac ctctgggcta cgggacagcc gaattcgcca aacgcatcac cctcggccgc 660ctgtctgagc cagaagatgt cgccgcctgc gtctcttatc tcgccagccc ggattccgat 720tatatgaccg gtcaatcgct gctgatcgat ggcgggatgg tattcaatta a 771151680DNAEnterobacter aerogenes 15atggacaaac agtatccgca gcgccagtgg gcgcacggcg ccgatctggt cgtcagccaa 60ctggaagcgc aaggcgtacg gcaggtcttc gggatccccg gcgctaaaat cgataaggtt 120ttcgactcgt tgctggactc ctcaatccgc attattccgg tacgtcacga ggccaacgcc 180gcctttatgg ccgccgcggt cgggcgcatt accggcaaag cgggcgtcgc gctggtgacc 240tccggacccg gttgttccaa cctgataacc gggatggcca ccgccaatag cgaaggcgac 300ccggtggtgg cgctgggcgg cgcggtcaaa cgcgcggata aagccaaaca ggtacaccag 360agtatggaca cggtggcgat gttcagcccg gtcaccaaat acgcggtaga agtgacctcg 420ccggatgcgc tggcggaagt ggtttctaac gcttttcgcg ccgccgagca gggtcgcccg 480ggcagcgcct tcgtcagtct gccgcaggat gtggtcgatg gtccggtgac cggcaaagtc 540ctgcccgcca gcagcgcgcc gcagatgggc gccgcgcctg acgaggcaat caatcaggtt 600gcgaagttga ttgcccaggc gaagaatccg gtgttcctgc ttggattaat ggccagccag 660acggaaaaca gcgccgcgct gcatcgtttg ctggaaacca gccatattcc ggtcaccagc 720acctatcagg ccgccggggc ggtcaatcag gataacttct cgcgcttcgc cgggcgcgtc 780gggctgttta acaatcaggc cggtgaccgc ttattgcaac tggccgacct ggttatctgc 840atcggctata gcccggtgga atacgaaccg gcgatgtgga acagcggcaa cgcgacgctg 900gtacatatcg acgtactgcc cgcctatgaa gagcgtaact acacgccgga tgtcgagctg 960gtgggcgaca tcgccggcac gctgaacaag ctggcgcaaa atatcgatca tcggctggtg 1020ctctcgccgc aggctgctga aatcctccac gaccgccagc atcagcggga actgcttgac 1080cgccgcggag cgcagttgaa tcagtttgcc ctgcacccgc tgcgtatcgt tcgcgccatg 1140caggatatcg tcaacagcga cgtcacgctg acggtcgata tggggagctt ccatatctgg 1200atcgcccgct atctctacag cttccgcgcc cgtcaggtga tgatctccaa cggtcagcag 1260accatgggcg tcgccctgcc gtgggccatc ggggcctggc tggtcaatcc gcagcgcaaa 1320gtggtctcgg tctccggcga tggcggtttt ctgcaatcca gcatggagct ggaaacggcg 1380gtccgcctga aagccaacat cctgcatctt atctgggtcg ataacggcta caacatggtc 1440gccatccagg aagagaaaaa atatcaacgc ctgtccggcg tcgagttcgg tcctatggat 1500tttaaagcct atgccgaatc cttcggcgcg aaagggtttg cggtggaaag cgctgaggcg 1560ctggagccga cgctacgcgc ggcgatggac gtcgacggcc cggcggtggt cgccatcccc 1620gtggattacc gtgataaccc gctgctgatg ggccagctac acctgagtca aattctttaa 168016873DNAUnknownVibrio sp. dhg 16atggctcacg gctgcgaagt cgtctgtgca tttgttaacg atgatctgtc agaacccgtt 60ttaaaacagc tatctcaggg cgggactaag cttatagcga tgcgttgtgc gggatttgat 120aaggtcgacc aacaagcggc taagaagcta ggcttgcaag tagtacatgt gcccgcctat 180tcacctgaag cggttgccga acatacggtt ggtatgatga tgtgtctaaa ccgtcgacta 240cacaaagcct atcagcgaac ccgggatgcg aatttctctc tggaaggttt agttggcttc 300aacttttttg gtaagactgt aggggtaata ggtacgggaa aaatcggcat tgctgcgatg 360agaattttta agggattagg catggaaatt ctttgccatg atccttacga aaacccactg 420gcaatagaga tgggcgcacg ttactgctct cttgaagata tatacgccaa tgcggatatt 480attactttgc attgcccgat gagtaaagaa aactaccacc tcctcaatgc cgactcattt 540tcgaaaatga aagatggggt gatgatcatt aatacgagtc gtggagagct attggattct 600gtagcagcaa tcgaagcgct aaaacaaggc agaatcggct cactaggctt ggatgtgtac 660gacaatgaaa aagagttgtt cttccaggat aaatcaaacg acattatcgt agacgatgtt 720ttccgccgac tgtcggcatg tcataacgtg ctgttcacgg gtcatcaagc tttcttaact 780cacgaagcgc tcaacaacat cgcgtcagtg acactaaata acgtagaagt attcttctct 840gggcaagttt caggcaatga actgatcaac taa 873171806DNAUnknownVibrio sp. dhg 17gtgcaaatta tcaccacaga tatcgcagtc atcggcgctg gcggcgctgg tcttcgtact 60gctattgcag cggcagaggc aaacccagat ttagaagtcg ctctgatttc taaagtttat 120cctatgcgct cacacacggt cgcagcggag ggtggctcag cagcagttat caaggatgaa 180gatagcttag ataaccactt caacgatacg gttggcggtg gcgactggct atgtgaacag 240gacgtcgttg aatattttgt agaaaacgca acccgcgaaa tgatccaaat ggaacaatgg 300ggttgtcctt ggagccgtaa agagaacggg gaagttaacg ttcgccgctt tggcggtatg 360aaggttgaac gaacttggtt tgcagcggat aaaaccggct tccatatgct tcacacccta 420ttccaaactt caatgaagta cagcaacatc aaacgttttg atgagtactt tgtgttggat 480ctgcttgttg acgatggtga agtacaaggt ctgatcgcca ttcacatgtc tgaaggtgag 540ttggtcacca tcaaagcgaa atctgttgtg cttgcgactg gtggcgcggg acgtgtttac 600cactgtaaca ccaatggcgg tatcgtaacg ggcgatggca tggcgatggc ttaccgtcac 660ggtgttccgc tacgtgatat ggaatttgtt caataccacc ctactggcct accaggtact 720ggtatcctaa tgacagaagg ttgtcgtggt gaaggtggta ttattgtcaa caaaaacggc 780taccgctacc tacaagacta cggcatgggc cctgaaactc cagtgggtca accgaaaaac 840aaatacatgg aactgggtcc acgtgacaaa gtttctcaag ctttctggca tgagcaacag 900aaaggcaaca ccatcaaaca cccactgggt gacgtggtgc acctagacct tcgccacctt 960ggtgaagagt acctacaaga acgtttacct ttcatctgtg agctagcaaa agcttacgtg 1020aacgttgatc ctgcaaaaga accaattcca attcgtccga ccgtgcacta caccatgggc 1080ggcatcgaaa ctgatggtgg ctgtgagact cgcgttaaag gtctattcgc agttggtgag 1140tgtgcgtcag ttggtctgca tggtgcgaac cgtcttggct ctaactctct ggctgagttc 1200gtggtatttg gccgagttgc gggtgaaaac gcagtgaaac gtgcagcaga attcaaaggc 1260tggaacgaca atgctatcgc agctcaagtg aaagctgttg aagaacgcat tgccagctta 1320atgaaccaag aaggcgatga aaactgggca gacatccgta ccgaaatggg ccacaccatg 1380gaagcgggtt gtggcatcta ccgccaagaa gatctgatgc aagcaaccat cgataagatc 1440acggaactta aacaacgtta caaacgcatt agcatcaaag acaaaggcaa agtgttcaac 1500actgaccttc tttacgcaat cgaagtcggt tacggcctag aagtggcaga agcgatggtt 1560cactctgcaa tcctgcgcaa agaatctcgc ggtgcacacc aacgtctcga tgatggctgc 1620actgaacgtg acgacgtgaa cttcctgaaa cactcacttg ctttctatca accagacgca 1680gcgcctagca tcgactacag caatgtaacc attactaagt ctcagcctaa agcgcgtcta 1740tacggtgaag ctgcagaaaa agccgcagca gaagaagcag cgaagaacgc agaggagcaa 1800gcataa 180618747DNAUnknownVibrio sp. dhg 18atgtcagcaa accgcatcca gaaagtagac attctgcgtt atgacccaga aaaagacgca 60gaaccgcact tacaaacttt cgaagtacca ttcgatgaaa ctatgtctgt gctcgacgcg 120attggttaca tcaaagataa cctagacaaa gacttatctt accgttggtc ttgtcgtatg 180gcgatctgtg gctcatgcgg catcatggtt aatggtgtgc ctaagctagc ttgtaagagc 240ttcttacgtg actaccaaaa tggtctgaaa atcgagccat tagcgaattt cccgattgag 300aaagacttga tcgttgatat gacgccattt atcgagcgtc ttgaagcgat caaaccttac 360atcattggta acgaccgtaa acctgaagac ggcacaaact tgcaaacgcc agagcaaatg 420gcgaagtaca agcagtttgc tggttgtatc aactgtggtc tgtgttacgc agcgtgtcct 480cagttcggtc tcaacccaga gttcatcggc ccggcagcgc taacattggc gcaccgttac 540aacttagaca gtcgtgataa cggtaaagct gagcgtatga agctgattaa cggtgagaat 600ggcgcctggg gttgtacgtt tgtaggttac tgttctgagg tttgtccaaa gagcgttgac 660cctgcagcag cagtaaacca aggcaaagta gagtcttcta tggacttcgt aattgcgatg 720ctgaaacctc aggaggcaga aggatga 74719384DNAUnknownVibrio sp. dhg 19atgagtaacc gtaaacctta cgttcgtgaa gtaaaacgca cttggtggaa ggaccatcct 60ttctaccgct tctacatgtt acgtgaagcg acggtactgc cactgattct attcaccatc 120ttcctgactt tcggcctggg ttcactagtg aaagggcctg aagcttggca aggctggtta 180gagttcatgg caaacccaat cgtagtcgcg atcaacatcg ttgcgctact tggaagcctg 240ttccacgcac aaaccttctt cagcatgatg ccacaggtga tgccaattcg cctaaaaggc 300aaacctgtgg gtaagaatat catcgtactg actcagtggg cagcggtcgc gtttatctca 360ctgatcgttc tcatcgtggt gtaa 38420378DNAUnknownVibrio sp. dhg 20atgaaaccaa attatagtgt aaacacagca ccaaaacgtt cagatgagcc aatctggtgg 60ggactgtttg gtgcaggcgg tacctggttt gcgatgatca ctcctatcac cgtacttgta 120cttggtatcc tcgttccact gggcgtgatt gatgcagacg ccatgagcta cgagcgagta 180tctgaattcg cgaccagtat cattggtgcg ctatttatca tcggtacact agcgctgcca 240atgtggcatg caatgcaccg tgttcaccac ggcatgcacg accttaagtt ccacactggt 300gtggtgggaa aagtggcatg ctatgcgttc gctggcctta tcagtgcgct atcagttatc 360tttatcttca tgatttaa 378212277DNAUnknownVibrio sp. dhg 21atggcagagc aatttgctaa agcttgggaa ggttttgctg caggtgattg gcaaaacgaa 60gtaaacgttc gtgatttcat tcagaagaac tacactccgt acgaaggcga cgaatctttc 120ctagtttctg aaggtactga agcaacaaac aagctttggg ctaaagtaat ggaaggtatc 180aaacaagaga acgcgactca cgctcctgtt gattttgata catctgttat ctctaccatc 240actgctcacg atgcaggcta catcgaaaaa gatcttgaaa ctatcgtagg tctacaaact 300gaagcgcctc taaaacgtgc gatcatccct aacggtggta ttcgcatggt tgaaggttca 360tgcaaagcat atgaccgcga actagaccca caagttaaga aaatcttcac agaataccgt 420aaaacacaca atgctggtgt tttcgatatc tacactcctg atatccttgc atgtcgtaag 480tctggtgtac taactggtct tcctgacgca tacggccgtg gtcgtatcat cggtgactac 540cgtcgcgttg cgctttacgg tatcgacttc ctaatgaagg acaaactagc tcagttcact 600tctctacaag agaaatttga gaacggcgaa gaccttcaca tgactatgca acttcgtgaa 660gaaattgcag agcagcaccg cgctctaggt caaatcaaac aaatggctgc gaaatacggt 720ttcgatattt ctcgccctgc tgaaactgca caagaagcta tccaatggac ttacttcggc 780tacctagctg ctgttaagtc tcaaaacggt gctgcaatgt ctctaggtcg tacttctaca 840ttcctagacg tgtacatcga gcgtgatatc gctgcaggta agatcactga agatcaagct 900caagaaatga tcgaccactt cgtaatgaaa ctacgtatgg ttcgtttcct acgtactcct 960gagtacgatg agctattctc tggcgaccca atttgggcaa cagaatcaat gggtggtatg 1020ggtcttgacg gtcgtacgct agtaacgcgt tctaacttcc gtttcctaaa cagcctatac 1080actatgggtc cttctccaga gccaaacatc actgttcttt ggtctgaagc acttccagat 1140ggtttcaaac gtttctgtgc aaaagtatct atcgatactt cttctatcca gtacgaaaac 1200gacgatctga tgcgtccaga catggaatca gacgattacg ctatcgcttg ttgtgtatct 1260ccaatggttg ttggtaagca aatgcagttc ttcggtgctc gtgcgaacct tgctaaaact 1320atgctttaca ccatcaacgg cggtatcgat gagaagctga agatccaagt tggtcctaag 1380atggacaaga tcgaaggtga atacctagat tacaacgagc tatgggaaaa aatggatcac 1440ttcatggatt ggttagctaa gcagtacgtg actgcactaa acagcatcca cttcatgcac 1500gacaagtaca gctacgaagc gtctctaatg gctctacatg accgtgacgt taaacgtaca 1560atggcttgtg gtatcgctgg tctatctgtt gctgctgact ctctatcagc aatcaaatac 1620gcgaaagtta aaccagttcg tgacgaagat ggtctagcaa tcgactttga aatcgaaggc 1680gattacccta aattcggtaa caacgacgct cgcgtagatg acatcgcttg tgaacttgtt 1740tctgtattta tgaacaagat ccgtgagctt aagacttacc gtgatgctat ccctactcag 1800tctatcctga ctatcacttc aaacgtggta tacggtaaga agactggtaa cacgcctgat 1860ggtcgtcgtg ctggtactcc atttgcgcca ggtgcaaacc caatgcacgg ccgtgatgag 1920aaaggtgcag tagcatcatt gacttcagta gcgaaactac cgtttgctga cgctcaagat 1980ggtatctctt acacattctc tatcgtgcca aatgcactag gtaaagaaga gactagccaa 2040cgtgctaacc ttgcaggcct aatggatggt tacttccacc acgaagctgg catcgaaggt 2100ggccaacacc taaacgtgaa cgtgcttaac cgcgaaactc tagaagacgc agttaaacac 2160ccagagaaat accctcagct aactatccgt gtatcgggtt acgctgtacg tttcaactct 2220ctgactgctg aacagcaagc tgacgttatc gctcgtacat tcactgaatc actataa 22772235DNAArtificial SequenceSynthetic 5' UTRmisc_feature(1)..(6)promotermisc_feature(26)..(29)promoter 22nnnnnngcta gctcagtcct aggkannnng ctagc 352335DNAArtificial Sequencepromoter 23cttatggcta gctcagtcct agggacagtg ctagc 352435DNAArtificial Sequencepromoter 24tttacggcta gctcagtcct agggatagtg ctagc 352535DNAArtificial Sequencepromoter 25ctgacggcta gctcagtcct agggatagtg ctagc 352635DNAArtificial Sequencepromoter 26ttgatggcta gctcagtcct agggattatg ctagc 352735DNAArtificial Sequencepromoter 27ttgatggcta gctcagtcct aggtacagtg ctagc 352835DNAArtificial Sequencepromoter 28ttgatggcta gctcagtcct aggtattgtg ctagc 352935DNAArtificial Sequencepromoter 29ttgatggcta gctcagtcct aggtactatg ctagc 353035DNAArtificial Sequencepromoter 30ttgacggcta gctcagtcct aggtactgtg ctagc 353135DNAArtificial Sequencepromoter 31ttgatggcta gctcagtcct aggtacaatg ctagc 353235DNAArtificial Sequencepromoter 32ttgatggcta gctcagtcct aggtatagtg ctagc 353335DNAArtificial Sequencepromoter 33ttgatggcta gctcagtcct aggtacaatg ctagc 353435DNAArtificial Sequencepromoter 34ttgatggcta gctcagtcct aggtataatg ctagc 353535DNAArtificial Sequencepromoter 35ttgacggcta gctcagtcct aggtacagtg ctagc 353625DNAArtificial

SequenceSynthetic 5' UTR 36acggagawtg ctyaaksagt csttt 253725DNAArtificial SequenceSynthetic 5' UTR 37acggagattg cttaagcagt cgttt 253825DNAArtificial SequenceSynthetic 5' UTR 38acggagaatg cttaatcagt cgttt 253925DNAArtificial SequenceSynthetic 5' UTR 39acggagattg cttaatcagt ccttt 254025DNAArtificial SequenceSynthetic 5' UTR 40acggagattg cttaagcagt cgttt 254125DNAArtificial SequenceSynthetic 5' UTR 41acggagaatg ctcaatgagt cgttt 254225DNAArtificial SequenceSynthetic 5' UTR 42acggagattg cttaatgagt cgttt 254325DNAArtificial SequenceSynthetic 5' UTR 43acggagaatg cttaatgagt cgttt 254425DNAArtificial SequenceSynthetic 5' UTR 44acggagaatg cttaatgagt cgttt 254525DNAArtificial SequenceSynthetic 5' UTR 45acggagattg cttaatgagt cgttt 254625DNAArtificial SequenceSynthetic 5' UTR 46acggagaatg ctcaaggagt cgttt 254725DNAArtificial SequenceSynthetic 5' UTR 47acggagattg cttaaggagt cgttt 254825DNAArtificial SequenceSynthetic 5' UTR 48acggagattg cttaaggagt ccttt 254925DNAArtificial SequenceSynthetic 5' UTR 49acggagaatg cttaaggagt cgttt 255025DNAArtificial SequenceSynthetic 5' UTR 50acggagattg cttaaggagt cgttt 25516455DNAArtificial SequencepACYCA_SXT 51gcactgatga gggtgtcagt gaagtgcttc atgtggcagg agaaaaaagg ctgcaccggt 60gcgtcagcag aatatgtgat acaggatata ttccgcttcc tcgctcactg actcgctacg 120ctcggtcgtt cgactgcggc gagcggaaat ggcttacgaa cggggcggag atttcctgga 180agatgccagg aagatactta acagggaagt gagagggccg cggcaaagcc gtttttccat 240aggctccgcc cccctgacaa gcatcacgaa atctgacgct caaatcagtg gtggcgaaac 300ccgacaggac tataaagata ccaggcgttt cccctggcgg ctccctcgtg cgctctcctg 360ttcctgcgaa aggacaagtt ttggtgactg cgctcctcca agccagttac ctcggttcaa 420agagttggta gctcagagaa ccttcgaaaa accgccctgc aaggcggttt tttcgttttc 480agagcaagag attacgcgca gaccaaaacg atctcaagaa gatcatctta ttaatcagat 540aaaatatttc tagatttcag tgcaatttat ctcttcaaat gtagcacctg aagtcagccc 600catacgatat aagttgtaat tctcatgtta gtcatgcccc gcgcccaccg gaaggagctg 660actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa 720cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 780ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt 840ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg 900agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat 960ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga 1020gatgtccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat 1080ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt 1140ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg 1200attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa 1260tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag 1320tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc 1380aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc 1440cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc 1500tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg 1560atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga 1620ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg 1680aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg 1740gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac 1800atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta 1860tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc 1920ccttttgaca attaatcatc ggctcgtata atgggaattg tgagcggata acaattaagg 1980agatatgcat ggatattaat actgaaactg agatcaagca aaagcattca ctaaccccct 2040ttcctgtttt cctaatcagc ccggcatttc gcgggcgata ttttcacagc tatttcagga 2100gttcagccat gaacgcttat tacattcagg atcgtcttga ggctcagagc tgggcgcgtc 2160actaccagca gctcgcccgt gaagagaaag aggcagaact ggcagacgac atggaaaaag 2220gcctgcccca gcacctgttt gaatcgctat gcatcgatca tttgcaacgc cacggggcca 2280gcaaaaaatc cattacccgt gcgtttgatg acgatgttga gtttcaggag cgcatggcag 2340aacacatccg gtacatggtt gaaaccattg ctcaccacca ggttgatatt gattcagagg 2400tataaaacga atggaaaaac caaagctaat tcaacgcttt gctgagcgct ttagtgtcga 2460tccaaacaag ttgttcgata ccctaaaagc aacagcattc aagcaacgtg acggtagtgc 2520accgaccaat gagcagatga tggcgctctt ggtggttgca gatcagtacg gcttgaaccc 2580tttcaccaaa gagatttttg cgttccctga taaacaagct gggattattc cagtggtagg 2640tgtcgatgga tggtctcgca tcattaatca acacgaccag tttgatggca tggagtttaa 2700gacttcagaa aacaaagtct ccctggatgg cgcgaaagaa tgcccggaat ggatggaatg 2760catcatctat cggcgcgacc gttcgcaccc agtcaaaatc actgaatacc tggatgaagt 2820ctatcgaccg ccttttgaag gtaacggcaa aaatggccct taccgggtag atggtccatg 2880gcagacgcac actaagcgaa tgctaagaca taaatccatg atccagtgtt cccgcattgc 2940gtttggcttt gtgggaattt tcgatcaaga cgaagcggag cgaattatcg aaggccaagc 3000aacacacgtt gttgagccat cggtgattcc acccgagcaa gttgatgatc gaacccgagg 3060gcttgtttac aagcttatcg agcgggcgga agcttcaaac gcttggaata gtgcattgga 3120atatgccaat gaacattttc aaggtgttga actgacgttt gcgaaacaag aaataattaa 3180tgcacagcaa caagcagcca aagcgctcac acagccttta gcttcttagc gccacgcatt 3240cattttacta accctggcgg gattattctc ccgtcagggg gaaggtctcg tctttttatt 3300ggagatcttc catgactaaa tcagcctcac tttttcgctt ggtattggtt gttgcccttg 3360tcttaggttc gattcaagcc ggtaaagcgg caattgattc ggttcaagca agtgttgttc 3420agcaccaaac agcgttagca caagctgcaa agtaaccact taaccctgaa ggggagttct 3480ctccttcagg ggagtctccc ttcaaaggag gcaatatgaa ggttatcgac ctatcacaac 3540gtactcctgc atggcaccag tggcgcattg caggggttac ggcatctgaa gccccaatta 3600ttatggggcg ttcaccctac aaaacacctt ggcgattatg ggcagaaaaa accggattcg 3660tattaccgga agacctatcg aataatccaa atgtgcttcg aggtatacgg ttggagcctc 3720aagcaaggcg agcatttgag aatgcgcata atgactttct tctgccgtta tgtgcagaag 3780ccgatcataa cgcaatcttt cgagccagct ttgatggcat caacgatgcg ggcgaacctg 3840ttgaactgaa atgtccttgc cagtcagttt ttgaggatgt gcaagctcac cgagaacaaa 3900gtgaggcgta ccagttgtat tgggtgcaag tacagcacca aatactggtc gccaatagca 3960cgcgtggttg gttggtattc tattttgagg atcaactgat tgagtttgaa atacaacgag 4020acgcggcgtt cttaactgag ttgcaagaaa cagcgcttca gttttgggag ttagtacaga 4080ccaaaaaaga accgtcaaaa tgccctgagc aagattgttt tgttcccaag ggtgaagccc 4140aataccgttg gacatcgcta tcacggcagt attgctcagc acatgccgaa gtggtccgac 4200tggaaaacca cattaaatct ttgaaagagg aaatgcgaga agctcagtcg aaattggtcg 4260ctatgatggg taactacgct catgccgact atgctggggt caaactcagc cgctacatga 4320tggcaggtac ggtggactat aagcaattgg ccaccgataa gttaggcgag ctggatgaac 4380aggttttagc tgcttaccga aaagcgccac aagagcggtt gcgcattagc accaataagc 4440cagagcagcc cgttgaaaca ccaatcaaaa tcagccttga gcaagagaac ttggttctgc 4500caggtgactc gccgagctca ttttactttt aacgcatcct cacgataata tccgggtagg 4560cgcaatcact ttcgtctact ccgttacaaa gcgaggctgg gtatttcccg gcctttctgt 4620tatccgaaat ccactgaaag cacagcggct ggctgaggag ataaataata aacgaggggc 4680tgtatgcaca aagcatcttc tgttgagtta agaacgagta tcgagatggc acatagcctt 4740gctcaaattg gaatcaggtt tgtgccaata ccagtagaaa cagacgaaga atcctttccg 4800cggccgccca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg 4860aggggttttt tgctgaaacc tcaggcattt gagaagcaca cggtcacact gcttccggta 4920gtcaataaac cggtaaacca gcaatagaca taagcggcta tttaacgacc ctgccctgaa 4980ccgacgacgg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 5040attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 5100ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 5160tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 5220aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc 5280acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 5340aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 5400agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 5460ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 5520agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 5580tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 5640tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 5700attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 5760taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 5820aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 5880caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 5940gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 6000cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 6060tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 6120acctgcatta tggtgaaagt tggaacctct tacgtgccga tcaacgtctc attttcgcca 6180aaagttggcc cagggcttcc cggtatcaac agggacacca ggatttattt attctgcgaa 6240gtgatcttcc gtcacaggta tttattcggc gcaaagtgcg tcgggtgatg ctgccaactt 6300actgatttag tgtatgatgg tgtttttgag gtgctccagt ggcttctgtt tctatcagct 6360gtccctcctg ttcagctact gacggggtgg tgcgtaacgg caaaagcacc gccggacatc 6420agcgctagcg gagtgtatac tggcttacta tgttg 6455523508DNAArtificial SequencepRSF_FLP 52cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc 60ggtcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg 120ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg 180ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct 240tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa 300catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc 360catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc 420catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt 480gaatatggct catactcttc ctttttcaat attattgaag catttatcag ggttattgtc 540tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggc atgcagcgct 600cttccgcttc ctcgctcact gactcgctac gctcggtcgt tcgactgcgg cgagcggtgt 660cagctcactc aaaagcggta atacggttat ccacagaatc aggggataaa gccggaaaga 720acatgtgagc aaaaagcaaa gcaccggaag aagccaacgc cgcaggcgtt tttccatagg 780ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gccagaggtg gcgaaacccg 840acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 900ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 960tctcatagct cacgctgttg gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 1020tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 1080gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccattgg taactgattt 1140agaggacttt gtcttgaagt tatgcacctg ttaaggctaa actgaaagaa cagattttgg 1200tgagtgcggt cctccaaccc acttaccttg gttcaaagag ttggtagctc agcgaacctt 1260gagaaaacca ccgttggtag cggtggtttt tctttattta tgagatgatg aatcaatcgg 1320tctatcaagt caacgaacag ctattccgtt actctagatt tcagtgcaat ttatctcttc 1380aaatgtagca cctgaagtca gccccatacg atataagttg taattctcat gttagtcatg 1440ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat 1500cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgcgagct cttgacggct 1560agctcagtcc taggtacagt gctagcatct cgcaaatcga aggagcctca tatgccacaa 1620tttggtatat tatgtaaaac accacctaag gtgcttgttc gtcagtttgt ggaaaggttt 1680gaaagacctt caggtgagaa aatagcatta tgtgctgctg aactaaccta tttatgttgg 1740atgattacac ataacggaac agcaatcaag agagccacat tcatgagcta taatactatc 1800ataagcaatt cgctgagttt cgatattgtc aataaatcac tccagtttaa atacaagacg 1860caaaaagcaa caattctgga agcctcatta aagaaattga ttcctgcttg ggaatttaca 1920attattcctt actatggaca aaaacatcaa tctgatatca ctgatattgt aagtagtttg 1980caattacagt tcgaatcatc ggaagaagca gataagggaa atagccacag taaaaaaatg 2040cttaaagcac ttctaagtga gggtgaaagc atctgggaga tcactgagaa aatactaaat 2100tcgtttgagt atacttcgag atttacaaaa acaaaaactt tataccaatt cctcttccta 2160gctactttca tcaattgtgg aagattcagc gatattaaga acgttgatcc gaaatcattt 2220aaattagtcc aaaataagta tctgggagta ataatccagt gtttagtgac agagacaaag 2280acaagcgtta gtaggcacat atacttcttt agcgcaaggg gtaggatcga tccacttgta 2340tatttggatg aatttttgag gaattctgaa ccagtcctaa aacgagtaaa taggaccggc 2400aattcttcaa gcaataaaca ggaataccaa ttattaaaag ataacttagt cagatcgtac 2460aataaagctt tgaagaaaaa tgcgccttat tcaatctttg ctataaaaaa tggcccaaaa 2520tctcacattg gaagacattt gatgacctca tttctttcaa tgaagggcct aacggagttg 2580actaatgttg tgggaaattg gagcgataag cgtgcttctg ccgtggccag gacaacgtat 2640actcatcaga taacagcaat acctgatcac tacttcgcac tagtttctcg gtactatgca 2700tatgatccaa tatcaaagga aatgatagca ttgaaggatg agactaatcc aattgaggag 2760tggcagcata tagaacagct aaagggtagt gctgaaggaa gcatacgata ccccgcatgg 2820aatgggataa tatcacagga ggtactagac tacctttcat cctacataaa tagacgcata 2880taagcggccg cccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt 2940cttgaggggt tttttgctga aacctcaggc atttgagaag cacacggtca cactgcttcc 3000ggtagtcaat aaaccggtaa accagcaata gacataagcg gctatttaac gaccctgccc 3060tgaaccgacg acaagctgac gaccgggtct ccgcaagtgg cacttttcgg ggaaatgtgc 3120gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt 3180aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta 3240tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag 3300ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata 3360caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg 3420acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca 3480ggccagccat tacgctcgtc atcaaaat 3508538798DNAArtificial SequencepACYC_idi_ispA_crtEBI 53tatagtgagt cgtattaatt tcctaatgca ggagtcgcat aagggagagc gtcgagatcc 60cggacaccat cgaatggcgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga 120gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg 180gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa 240cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac 300aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc 360acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg 420tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc 480ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca 540ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga 600cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc 660tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg 720cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag 780cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga 840atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa 900tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga catctcggta gtgggatacg 960acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc 1020gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga 1080agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata 1140cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt 1200cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag 1260gcaccgggat ctcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg 1320gcgcggggca tgactaacat gagaattaca acttatatcg tatggggctg acttcaggtg 1380ctacatttga agagataaat tgcactgaaa tctagagcgg ttcagtagaa aagatcaaag 1440gatcttcttg agatcctttt tttctgcgcg taatcttttg ccctgtaaac gaaaaaacca 1500cctggggagg tggtttgatc gaaggttaag tcagttgggg aactgcttaa ccgtggtaac 1560tggctttcgc agagcacagc aaccaaatct gtccttccag tgtagccgga ctttggcgca 1620cacttcaaga gcaaccgcgt gtttagctaa acaaatcctc tgcgaactcc cagttaccaa 1680tggctgctgc cagtggcgtt ttaccgtgct tttccgggtt ggactcaagt gaacagttac 1740cggataaggc gcagcagtcg ggctgaacgg ggagttcttg cttacagccc agcttggagc 1800gaacgaccta caccgagccg agataccagt gtgtgagcta tgagaaagcg ccacacttcc 1860cgtaagggag aaaggcggaa caggtatccg gtaaacggca gggtcggaac aggagagcgc 1920aagagggagc gacccgccgg aaacggtggg gatctttaag tcctgtcggg tttcgcccgt 1980actgtcagat tcatggttga gcctcacggc tcccacagat gcaccggaaa agcgtctgtt 2040tatgtgaact ctggcaggag ggcggagcct atggaaaaac gccaccggcg cggccctgct 2100gttttgcctc acatgttagt cccctgctta tccacggaat ctgtgggtaa ctttgtatgt 2160gtccgcagcg cccgccgcag tctcacgccc ggagcgtagc gaccgagtga gctagctatt 2220tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa 2280atgcttcaat aatattgaaa aaggaagagt atgagggaag cggtgatcgc cgaagtatcg 2340actcaactat cagaggtagt tggcgtcatc gagcgccatc tcgaaccgac gttgctggcc 2400gtacatttgt acggctccgc agtggatggc ggcctgaagc cacacagtga tattgatttg 2460ctggttacgg tgaccgtaag gcttgatgaa acaacgcggc gagctttgat caacgacctt 2520ttggaaactt cggcttcccc tggagagagc gagattctcc gcgctgtaga agtcaccatt 2580gttgtgcacg acgacatcat tccgtggcgt tatccagcta agcgcgaact gcaatttgga 2640gaatggcagc gcaatgacat tcttgcaggt atcttcgagc cagccacgat cgacattgat 2700ctggctatct tgctgacaaa agcaagagaa catagcgttg ccttggtagg tccagcggcg 2760gaggaactct ttgatccggt tcctgaacag gatctatttg aggcgctaaa tgaaacctta 2820acgctatgga actcgccgcc cgactgggct ggcgatgagc gaaatgtagt gcttacgttg 2880tcccgcattt ggtacagcgc agtaaccggc aaaatcgcgc cgaaggatgt cgctgccgac 2940tgggcaatgg agcgcctgcc ggcccagtat cagcccgtca tacttgaagc tagacaggct 3000tatcttggac aagaagaaga tcgcttggcc tcgcgcgcag atcagttgga agaatttgtc 3060cactacgtga aaggcgagat caccaaggta gtcggcaaat aatgtctaac aattcgttca 3120agccgagggg ccgcaagatc cggccacgat gacccggtcg tcggttcagg gcagggtcgt 3180taaatagccg cttatgtcta ttgctggttt accggtttat tgactaccgg aagcagtgtg 3240accgtgtgct tctcaaatgc ctgaggtttc agcaaaaaac ccctcaagac ccgtttagag 3300gccccaaggg gttatgctag ttattgctca gcggtggcag cagcctaggt taattaagct 3360gcgctagtag acgagtccat gtgctggcgt tcaaatttcg cagcagcggt ttctttacca 3420gactcgaggg

taccgacgtc agcgatcgcg tggccggccg atatccaatt gagatctttg 3480acggctagct cagtcctagg tacagtgcta gccatcaaca aataaggagg agctacaatg 3540cagaccgaac atgtgattct gctgaacgcg cagggcgtgc cgaccggcac cctggaaaaa 3600tatgcggcgc ataccgcgga tacccgcctg catctggcgt ttagcagctg gctgtttaac 3660gcgaaaggcc agctgctggt gacccgccgc gcgctgagca aaaaagcgtg gccgggcgtg 3720tggaccaaca gcgtgtgcgg ccatccgcag ctgggcgaaa gcaacgaaga tgcggtgatt 3780cgccgctgcc gctatgaact gggcgtggaa attaccccgc cggaaagcat ttatccggat 3840tttcgctatc gcgcgaccga tccgagcggc attgtggaaa acgaagtgtg cccggtgttt 3900gcggcgcgca ccaccagcgc gctgcagatt aacgatgatg aagtgatgga ttatcagtgg 3960tgcgatctgg cggatgtgct gcatggcatt gatgcgaccc cgtgggcgtt tagcccgtgg 4020atggtgatgc aggcgaccaa ccgcgaagcg cgcaaacgcc tgagcgcgtt tacccagctg 4080aaataaaaaa accccgacat ttgccggggt tgtgagcgcc gcaaaccccg cttcggcggg 4140gtttcgccgc agatctttga cggctagctc agtcctaggt acagtgctag ccacgaccat 4200ataaggagga tagaagatgg atttcccgca gcagctggaa gcgtgtgtga aacaggcgaa 4260ccaggcgctg agccgcttta ttgcgccgct gccgtttcag aacaccccgg tggtggaaac 4320catgcagtat ggcgcgctgc tgggcggcaa acgcctgcgc ccgtttctgg tgtatgcgac 4380cggccacatg tttggcgtga gcaccaacac cctggatgcg ccggcggcgg cggtggaatg 4440cattcatgcg tatagcctga ttcatgatga tctgccggcg atggatgatg atgatctgcg 4500ccgcggcctg ccgacctgcc atgtgaaatt tggcgaagcg aacgcgattc tggcgggcga 4560tgcgctgcag accctggcgt ttagcattct gagcgatgcg gatatgccgg aagtgagcga 4620tcgcgatcgc attagcatga ttagcgaact ggcgagcgcg agcggcattg cgggcatgtg 4680cggcggccag gcgctggatc tggatgcgga aggcaaacat gtgccgctgg atgcgctgga 4740acgcattcat cgccataaaa ccggcgcgct gattcgcgcg gcggtgcgcc tgggcgcgct 4800gagcgcgggc gataaaggcc gccgcgcgct gccggtgctg gataaatatg cggaaagcat 4860tggcctggcg tttcaggtgc aggatgatat tctggatgtg gtgggcgata ccgcgaccct 4920gggcaaacgc cagggcgcgg atcagcagct gggcaaaagc acctatccgg cgctgctggg 4980cctggaacag gcgcgcaaaa aagcgcgcga tctgattgat gatgcgcgcc agagcctgaa 5040acagctggcg gaacagagcc tggataccag cgcgctggaa gcgctggcgg attatattat 5100tcagcgcaac aaataataaa caataagtat taataggccc ctgatcgccg caaaccccgc 5160ttcggcgggg tttcgccgca agcttttgac ggctagctca gtcctaggta cagtgctagc 5220taacaaaaca aaaggaggta atagaatggt atctggctca aaggctggcg tctcgccaca 5280tcgcgaaatt gaagtgatgc gccagagcat tgatgatcat ctggcgggcc tgctgccgga 5340aaccgatagc caggatattg tgagcctggc gatgcgcgaa ggcgtgatgg cgccgggcaa 5400acgcattcgc ccgctgctga tgctgctggc ggcgcgcgat ctgcgctatc agggcagtat 5460gccgaccctg ctggatctgg cgtgcgcggt ggaactgacc cataccgcga gcctgatgct 5520ggatgatatg ccgtgcatgg ataacgcgga actgcgccgc ggccagccga ccacccataa 5580aaaatttggc gaaagcgtgg cgattctggc gagcgtgggc ctgctgagca aagcgtttgg 5640cctgattgcg gcgaccggcg atctgccggg cgaacgccgc gcgcaggcgg tgaacgaact 5700gagcaccgcg gtgggcgtgc agggcctggt gctgggccag tttcgcgatc tgaacgatgc 5760ggcgctggat cgcaccccgg atgcgattct gagcaccaac catctgaaaa ccggcattct 5820gtttagcgcg atgctgcaga ttgtggcgat tgcgagcgcg agcagcccga gcacccgcga 5880aaccctgcac gcgtttgcgc tggattttgg ccaggcgttt cagctgctgg atgatctgcg 5940cgatgatcat ccggaaaccg gcaaagatcg caacaaagat gcgggcaaaa gcaccctggt 6000gaaccgcctg ggcgcggatg cggcgcgcca gaaactgcgc gaacatattg atagcgcgga 6060taaacatctg acctttgcgt gcccgcaggg cggcgcgatt cgccagttta tgcatctgtg 6120gtttggccat catctggcgg attggagccc ggtgatgaaa attgcgtaat accgcccttt 6180tgggttcaag cagtacataa cctaatatcc taaaggagcg aaaactatgt cccaaccccc 6240cttgctagac cacgcaaccc agacgatggc gaacggcagc aagagctttg cgaccgcggc 6300gaaactgttt gatccggcga cccgccgcag cgtgctgatg ctgtatacct ggtgccgcca 6360ttgcgatgat gtgattgatg atcagacgca tggctttgcg agcgaagcgg cggcggaaga 6420agaagcgacc cagcgcctgg cgcgcctgcg caccctgacc ctggcggcgt ttgaaggcgc 6480ggaaatgcag gacccggcgt ttgcggcgtt tcaggaagtg gcgctgaccc acggcattac 6540cccgcgcatg gcgctggatc atctggatgg ctttgcgatg gatgtggcgc agacccgcta 6600tgtgaccttt gaagataccc tgcgctattg ctatcatgtg gcgggcgtgg tgggcctgat 6660gatggcgcgc gtgatgggcg tgcgcgatga acgcgtgctg gatcgcgcgt gcgatctggg 6720cctggcgttt cagctgacca acattgcgcg cgatattatt gatgatgcgg cgattgatcg 6780ctgctatctg ccggcggaat ggctgcagga tgcgggcctg accccggaaa actatgcggc 6840gcgcgaaaac cgcgcggcgc tggcgcgcgt ggcggaacgc ctgattgatg cggcggaacc 6900gtattatatt agcagccagg cgggcctgca tgatctgccg ccgcgctgcg cgtgggcgat 6960tgcgaccgcg cgcagcgtgt atcgcgaaat tggcattaaa gtgaaagcgg cgggcggcag 7020cgcgtgggat cgccgccagc ataccagcaa aggcgaaaaa attgcgatgc tgatggcggc 7080gccgggccag gtgattcgcg cgaaaaccac ccgcgtgacc ccgcgcccgg cgggcctgtg 7140gcagcgcccg gtgtaagcgg gcggccatga cgttcacgca ggatcgatag agtcaacaag 7200gagttattat catgaaaaaa acggttgtga tcggcgctgg gttcggcggc ctggcgctgg 7260cgattcgcct gcaggcggcg ggcattccga ccgtgctgct ggaacagcgc gataaaccgg 7320gcggccgcgc gtatgtgtgg catgatcagg gctttacctt tgatgcgggc ccgaccgtga 7380ttaccgatcc gaccgcgctg gaagcgctgt ttaccctggc gggccgccgc atggaagatt 7440atgtgcgcct gctgccggtg aaaccgtttt atcgcctgtg ctgggaaagc ggcaaaaccc 7500tggattatgc gaacgatagc gcggaactgg aagcgcagat tacccagttt aacccgcgcg 7560atgtggaagg ctatcgccgc tttctggcgt atagccaggc ggtgtttcag gaaggctatc 7620tgcgcctggg cagcgtgccg tttctgagct ttcgcgatat gctgcgcgcg ggcccgcagc 7680tgctgaaact gcaggcgtgg cagagcgtgt atcagagcgt gagccgcttt attgaagatg 7740aacatctgcg ccaggcgttt agctttcata gcctgctggt gggcggcaac ccgtttacca 7800ccagcagcat ttataccctg attcatgcgc tggaacgcga atggggcgtg tggtttccgg 7860aaggcggcac cggcgcgctg gtgaacggca tggtgaaact gtttaccgat ctgggcggcg 7920aaattgaact gaacgcgcgc gtggaagaac tggtggtggc ggataaccgc gtgagccagg 7980tgcgcctggc ggatggccgc atttttgata ccgatgcggt ggcgagcaac gcggatgtgg 8040tgaacaccta taaaaaactg ctgggccatc atccggtggg ccagaaacgc gcggcggcgc 8100tggaacgcaa aagcatgagc aacagcctgt ttgtgctgta ttttggcctg aaccagccgc 8160atagccagct ggcgcatcat accatttgct ttggcccgcg ctatcgcgaa ctgattgatg 8220aaatttttac cggcagcgcg ctggcggatg attttagcct gtatctgcat agcccgtgcg 8280tgaccgatcc gagcctggcg ccgccgggct gcgcgagctt ttatgtgctg gcgccggtgc 8340cgcatctggg caacgcgccg ctggattggg cgcaggaagg cccgaaactg cgcgatcgca 8400tttttgatta tctggaagaa cgctatatgc cgggcctgcg cagccagctg gtgacccagc 8460gcatttttac cccggcggat tttcatgata ccctggatgc gcatctgggc agcgcgttta 8520gcattgaacc gctgctgacc cagagcgcgt ggtttcgccc gcataaccgc gatagcgata 8580ttgcgaacct gtatctggtg ggcgcgggca cccatccggg cgcgggcatt ccgggcgtgg 8640tggcgagcgc gaaagcgacc gcgagcctga tgatcgaaga cctgcagtaa cgccgcaaac 8700cccgcttcgg cggggtttcg ccgcccatgg tatatctcct tattaaagtt aaacaaaatt 8760atttctacag gggaattgtt atccgctcac aattcccc 87985410842DNAArtificial SequencepACYC_idi_ispA_crtEBI_dxs 54ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag 60gagatatacc atgggcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga 120ctcctgcatt aggaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc 180ccctgtagaa ataattttgt ttaactttaa taaggagata taccattgag atctttgacg 240gctagctcag tcctaggtac agtgctagcc atcaacaaat aaggaggagc tacaatgcag 300accgaacatg tgattctgct gaacgcgcag ggcgtgccga ccggcaccct ggaaaaatat 360gcggcgcata ccgcggatac ccgcctgcat ctggcgttta gcagctggct gtttaacgcg 420aaaggccagc tgctggtgac ccgccgcgcg ctgagcaaaa aagcgtggcc gggcgtgtgg 480accaacagcg tgtgcggcca tccgcagctg ggcgaaagca acgaagatgc ggtgattcgc 540cgctgccgct atgaactggg cgtggaaatt accccgccgg aaagcattta tccggatttt 600cgctatcgcg cgaccgatcc gagcggcatt gtggaaaacg aagtgtgccc ggtgtttgcg 660gcgcgcacca ccagcgcgct gcagattaac gatgatgaag tgatggatta tcagtggtgc 720gatctggcgg atgtgctgca tggcattgat gcgaccccgt gggcgtttag cccgtggatg 780gtgatgcagg cgaccaaccg cgaagcgcgc aaacgcctga gcgcgtttac ccagctgaaa 840taaaaaaacc ccgacatttg ccggggttgt gagcgccgca aaccccgctt cggcggggtt 900tcgccgcaga tctttgacgg ctagctcagt cctaggtaca gtgctagcca cgaccatata 960aggaggatag aagatggatt tcccgcagca gctggaagcg tgtgtgaaac aggcgaacca 1020ggcgctgagc cgctttattg cgccgctgcc gtttcagaac accccggtgg tggaaaccat 1080gcagtatggc gcgctgctgg gcggcaaacg cctgcgcccg tttctggtgt atgcgaccgg 1140ccacatgttt ggcgtgagca ccaacaccct ggatgcgccg gcggcggcgg tggaatgcat 1200tcatgcgtat agcctgattc atgatgatct gccggcgatg gatgatgatg atctgcgccg 1260cggcctgccg acctgccatg tgaaatttgg cgaagcgaac gcgattctgg cgggcgatgc 1320gctgcagacc ctggcgttta gcattctgag cgatgcggat atgccggaag tgagcgatcg 1380cgatcgcatt agcatgatta gcgaactggc gagcgcgagc ggcattgcgg gcatgtgcgg 1440cggccaggcg ctggatctgg atgcggaagg caaacatgtg ccgctggatg cgctggaacg 1500cattcatcgc cataaaaccg gcgcgctgat tcgcgcggcg gtgcgcctgg gcgcgctgag 1560cgcgggcgat aaaggccgcc gcgcgctgcc ggtgctggat aaatatgcgg aaagcattgg 1620cctggcgttt caggtgcagg atgatattct ggatgtggtg ggcgataccg cgaccctggg 1680caaacgccag ggcgcggatc agcagctggg caaaagcacc tatccggcgc tgctgggcct 1740ggaacaggcg cgcaaaaaag cgcgcgatct gattgatgat gcgcgccaga gcctgaaaca 1800gctggcggaa cagagcctgg ataccagcgc gctggaagcg ctggcggatt atattattca 1860gcgcaacaaa taataaacaa taagtattaa taggcccctg atcgccgcaa accccgcttc 1920ggcggggttt cgccgcaagc ttttgacggc tagctcagtc ctaggtacag tgctagctaa 1980caaaacaaaa ggaggtaata gaatggtatc tggctcaaag gctggcgtct cgccacatcg 2040cgaaattgaa gtgatgcgcc agagcattga tgatcatctg gcgggcctgc tgccggaaac 2100cgatagccag gatattgtga gcctggcgat gcgcgaaggc gtgatggcgc cgggcaaacg 2160cattcgcccg ctgctgatgc tgctggcggc gcgcgatctg cgctatcagg gcagtatgcc 2220gaccctgctg gatctggcgt gcgcggtgga actgacccat accgcgagcc tgatgctgga 2280tgatatgccg tgcatggata acgcggaact gcgccgcggc cagccgacca cccataaaaa 2340atttggcgaa agcgtggcga ttctggcgag cgtgggcctg ctgagcaaag cgtttggcct 2400gattgcggcg accggcgatc tgccgggcga acgccgcgcg caggcggtga acgaactgag 2460caccgcggtg ggcgtgcagg gcctggtgct gggccagttt cgcgatctga acgatgcggc 2520gctggatcgc accccggatg cgattctgag caccaaccat ctgaaaaccg gcattctgtt 2580tagcgcgatg ctgcagattg tggcgattgc gagcgcgagc agcccgagca cccgcgaaac 2640cctgcacgcg tttgcgctgg attttggcca ggcgtttcag ctgctggatg atctgcgcga 2700tgatcatccg gaaaccggca aagatcgcaa caaagatgcg ggcaaaagca ccctggtgaa 2760ccgcctgggc gcggatgcgg cgcgccagaa actgcgcgaa catattgata gcgcggataa 2820acatctgacc tttgcgtgcc cgcagggcgg cgcgattcgc cagtttatgc atctgtggtt 2880tggccatcat ctggcggatt ggagcccggt gatgaaaatt gcgtaatacc gcccttttgg 2940gttcaagcag tacataacct aatatcctaa aggagcgaaa actatgtccc aacccccctt 3000gctagaccac gcaacccaga cgatggcgaa cggcagcaag agctttgcga ccgcggcgaa 3060actgtttgat ccggcgaccc gccgcagcgt gctgatgctg tatacctggt gccgccattg 3120cgatgatgtg attgatgatc agacgcatgg ctttgcgagc gaagcggcgg cggaagaaga 3180agcgacccag cgcctggcgc gcctgcgcac cctgaccctg gcggcgtttg aaggcgcgga 3240aatgcaggac ccggcgtttg cggcgtttca ggaagtggcg ctgacccacg gcattacccc 3300gcgcatggcg ctggatcatc tggatggctt tgcgatggat gtggcgcaga cccgctatgt 3360gacctttgaa gataccctgc gctattgcta tcatgtggcg ggcgtggtgg gcctgatgat 3420ggcgcgcgtg atgggcgtgc gcgatgaacg cgtgctggat cgcgcgtgcg atctgggcct 3480ggcgtttcag ctgaccaaca ttgcgcgcga tattattgat gatgcggcga ttgatcgctg 3540ctatctgccg gcggaatggc tgcaggatgc gggcctgacc ccggaaaact atgcggcgcg 3600cgaaaaccgc gcggcgctgg cgcgcgtggc ggaacgcctg attgatgcgg cggaaccgta 3660ttatattagc agccaggcgg gcctgcatga tctgccgccg cgctgcgcgt gggcgattgc 3720gaccgcgcgc agcgtgtatc gcgaaattgg cattaaagtg aaagcggcgg gcggcagcgc 3780gtgggatcgc cgccagcata ccagcaaagg cgaaaaaatt gcgatgctga tggcggcgcc 3840gggccaggtg attcgcgcga aaaccacccg cgtgaccccg cgcccggcgg gcctgtggca 3900gcgcccggtg taagcgggcg gccatgacgt tcacgcagga tcgatagagt caacaaggag 3960ttattatcat gaaaaaaacg gttgtgatcg gcgctgggtt cggcggcctg gcgctggcga 4020ttcgcctgca ggcggcgggc attccgaccg tgctgctgga acagcgcgat aaaccgggcg 4080gccgcgcgta tgtgtggcat gatcagggct ttacctttga tgcgggcccg accgtgatta 4140ccgatccgac cgcgctggaa gcgctgttta ccctggcggg ccgccgcatg gaagattatg 4200tgcgcctgct gccggtgaaa ccgttttatc gcctgtgctg ggaaagcggc aaaaccctgg 4260attatgcgaa cgatagcgcg gaactggaag cgcagattac ccagtttaac ccgcgcgatg 4320tggaaggcta tcgccgcttt ctggcgtata gccaggcggt gtttcaggaa ggctatctgc 4380gcctgggcag cgtgccgttt ctgagctttc gcgatatgct gcgcgcgggc ccgcagctgc 4440tgaaactgca ggcgtggcag agcgtgtatc agagcgtgag ccgctttatt gaagatgaac 4500atctgcgcca ggcgtttagc tttcatagcc tgctggtggg cggcaacccg tttaccacca 4560gcagcattta taccctgatt catgcgctgg aacgcgaatg gggcgtgtgg tttccggaag 4620gcggcaccgg cgcgctggtg aacggcatgg tgaaactgtt taccgatctg ggcggcgaaa 4680ttgaactgaa cgcgcgcgtg gaagaactgg tggtggcgga taaccgcgtg agccaggtgc 4740gcctggcgga tggccgcatt tttgataccg atgcggtggc gagcaacgcg gatgtggtga 4800acacctataa aaaactgctg ggccatcatc cggtgggcca gaaacgcgcg gcggcgctgg 4860aacgcaaaag catgagcaac agcctgtttg tgctgtattt tggcctgaac cagccgcata 4920gccagctggc gcatcatacc atttgctttg gcccgcgcta tcgcgaactg attgatgaaa 4980tttttaccgg cagcgcgctg gcggatgatt ttagcctgta tctgcatagc ccgtgcgtga 5040ccgatccgag cctggcgccg ccgggctgcg cgagctttta tgtgctggcg ccggtgccgc 5100atctgggcaa cgcgccgctg gattgggcgc aggaaggccc gaaactgcgc gatcgcattt 5160ttgattatct ggaagaacgc tatatgccgg gcctgcgcag ccagctggtg acccagcgca 5220tttttacccc ggcggatttt catgataccc tggatgcgca tctgggcagc gcgtttagca 5280ttgaaccgct gctgacccag agcgcgtggt ttcgcccgca taaccgcgat agcgatattg 5340cgaacctgta tctggtgggc gcgggcaccc atccgggcgc gggcattccg ggcgtggtgg 5400cgagcgcgaa agcgaccgcg agcctgatga tcgaagacct gcagtaacgc cgcaaacccc 5460gcttcggcgg ggtttcgccg cccatgaatt ggatatcggc cggccacgcg atcgctgacg 5520tcggtacctt gacggctagc tcagtcctag gtacagtgct agccgtctcc acataaggag 5580cagttcacat gagttttgat attgccaaat acccgaccct ggcactggtc gactccaccc 5640aggagttacg actgttgccg aaagagagtt taccgaaact ctgcgacgaa ctgcgccgct 5700atttactcga cagcgtgagc cgttccagcg ggcacttcgc ctccgggctg ggcacggtcg 5760aactgaccgt ggcgctgcac tatgtctaca acaccccgtt tgaccaattg atttgggatg 5820tggggcatca ggcttatccg cataaaattt tgaccggacg ccgcgacaaa atcggcacca 5880tccgtcagaa aggcggtctg cacccgttcc cgtggcgcgg cgaaagcgaa tatgacgtat 5940taagcgtcgg gcattcatca acctccatca gtgccggaat tggtattgcg gttgctgccg 6000aaaaagaagg caaaaatcgc cgcaccgtct gtgtcattgg cgatggcgcg attaccgcag 6060gcatggcgtt tgaagcgatg aatcacgcgg gcgatatccg tcctgatatg ctggtgattc 6120tcaacgacaa tgaaatgtcg atttccgaaa atgtcggcgc gctcaacaac catctggcac 6180agctgctttc cggtaagctt tactcttcac tgcgcgaagg cgggaaaaaa gttttctctg 6240gcgtgccgcc aattaaagag ctgctcaaac gcaccgaaga acatattaaa ggcatggtag 6300tgcctggcac gttgtttgaa gagctgggct ttaactacat cggcccggtg gacggtcacg 6360atgtgctggg gcttatcacc acgctaaaga acatgcgcga cctgaaaggc ccgcagttcc 6420tgcatatcat gaccaaaaaa ggtcgtggtt atgaaccggc agaaaaagac ccgatcactt 6480tccacgccgt gcctaaattt gatccctcca gcggttgttt gccgaaaagt agcggcggtt 6540tgccgagcta ttcaaaaatc tttggcgact ggttgtgcga aacggcagcg aaagacaaca 6600agctgatggc gattactccg gcgatgcgtg aaggttccgg catggtcgag ttttcacgta 6660aattcccgga tcgctacttc gacgtggcaa ttgccgagca acacgcggtg acctttgctg 6720cgggtctggc gattggtggg tacaaaccca ttgtcgcgat ttactccact ttcctgcaac 6780gcgcctatga tcaggtgctg catgacgtgg cgattcaaaa gcttccggtc ctgttcgcca 6840tcgaccgcgc gggcattgtt ggtgctgacg gtcaaaccca tcagggtgct tttgatctct 6900cttacctgcg ctgcataccg gaaatggtca ttatgacccc gagcgatgaa aacgaatgtc 6960gccagatgct ctataccggc tatcactata acgatggccc gtcagcggtg cgctacccgc 7020gtggcaacgc ggtcggcgtg gaactgacgc cgctggaaaa actaccaatt ggcaaaggca 7080ttgtgaagcg tcgtggcgag aaactggcga tccttaactt tggtacgctg atgccagaag 7140cggcgaaagt cgccgaatcg ctgaacgcca cgctggtcga tatgcgtttt gtgaaaccgc 7200ttgatgaagc gttaattctg gaaatggccg ccagccatga agcgctggtc accgtagaag 7260aaaacgccat tatgggcggc gcaggcagcg gcgtgaacga agtgctgatg gcccatcgta 7320aaccagtacc cgtgctgaac attggcctgc cggacttctt tattccgcaa ggaactcagg 7380aagaaatgcg cgccgaactc ggcctcgatg ccgctggtat ggaagccaaa atcaaggcct 7440ggctggcata acgccgcaaa ccccgcttcg gcggggtttc gccgcttaat taacctaggc 7500tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag 7560gggttttttg ctgaaacctc aggcatttga gaagcacacg gtcacactgc ttccggtagt 7620caataaaccg gtaaaccagc aatagacata agcggctatt taacgaccct gccctgaacc 7680gacgaccggg tcatcgtggc cggatcttgc ggcccctcgg cttgaacgaa ttgttagaca 7740ttatttgccg actaccttgg tgatctcgcc tttcacgtag tggacaaatt cttccaactg 7800atctgcgcgc gaggccaagc gatcttcttc ttgtccaaga taagcctgtc tagcttcaag 7860tatgacgggc tgatactggg ccggcaggcg ctccattgcc cagtcggcag cgacatcctt 7920cggcgcgatt ttgccggtta ctgcgctgta ccaaatgcgg gacaacgtaa gcactacatt 7980tcgctcatcg ccagcccagt cgggcggcga gttccatagc gttaaggttt catttagcgc 8040ctcaaataga tcctgttcag gaaccggatc aaagagttcc tccgccgctg gacctaccaa 8100ggcaacgcta tgttctcttg cttttgtcag caagatagcc agatcaatgt cgatcgtggc 8160tggctcgaag atacctgcaa gaatgtcatt gcgctgccat tctccaaatt gcagttcgcg 8220cttagctgga taacgccacg gaatgatgtc gtcgtgcaca acaatggtga cttctacagc 8280gcggagaatc tcgctctctc caggggaagc cgaagtttcc aaaaggtcgt tgatcaaagc 8340tcgccgcgtt gtttcatcaa gccttacggt caccgtaacc agcaaatcaa tatcactgtg 8400tggcttcagg ccgccatcca ctgcggagcc gtacaaatgt acggccagca acgtcggttc 8460gagatggcgc tcgatgacgc caactacctc tgatagttga gtcgatactt cggcgatcac 8520cgcttccctc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct 8580catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagcta gctcactcgg 8640tcgctacgct ccgggcgtga gactgcggcg ggcgctgcgg acacatacaa agttacccac 8700agattccgtg gataagcagg ggactaacat gtgaggcaaa acagcagggc cgcgccggtg 8760gcgtttttcc ataggctccg ccctcctgcc agagttcaca taaacagacg cttttccggt 8820gcatctgtgg gagccgtgag gctcaaccat gaatctgaca gtacgggcga aacccgacag 8880gacttaaaga tccccaccgt ttccggcggg tcgctccctc ttgcgctctc ctgttccgac 8940cctgccgttt accggatacc tgttccgcct ttctccctta cgggaagtgt ggcgctttct 9000catagctcac acactggtat ctcggctcgg tgtaggtcgt tcgctccaag ctgggctgta 9060agcaagaact ccccgttcag cccgactgct gcgccttatc cggtaactgt tcacttgagt 9120ccaacccgga aaagcacggt aaaacgccac tggcagcagc cattggtaac tgggagttcg 9180cagaggattt gtttagctaa acacgcggtt gctcttgaag tgtgcgccaa agtccggcta 9240cactggaagg acagatttgg ttgctgtgct ctgcgaaagc cagttaccac ggttaagcag 9300ttccccaact gacttaacct tcgatcaaac cacctcccca ggtggttttt tcgtttacag 9360ggcaaaagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctactg 9420aaccgctcta gatttcagtg caatttatct cttcaaatgt agcacctgaa gtcagcccca 9480tacgatataa gttgtaattc tcatgttagt catgccccgc gcccaccgga aggagctgac 9540tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact 9600tacattaatt

gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct 9660gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg 9720tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag 9780agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg 9840tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga 9900tgtccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct 9960gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt 10020gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat 10080tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg 10140ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc 10200gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa 10260gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca 10320gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt 10380tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat 10440cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg 10500tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa 10560tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc 10620tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat 10680cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc 10740atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc 10800ttatgcgact cctgcattag gaaattaata cgactcacta ta 10842556875DNAArtificial SequencepACYC_BudACB 55tgcacgaacc ccccgttcag tccgaccgct gcgccttatc cggtaactat cgtcttgagt 60ccaacccgga aagacatgca aaagcaccac tggcagcagc cactggtaat tgatttagag 120gagttagtct tgaagtcatg cgccggttaa ggctaaactg aaaggacaag ttttggtgac 180tgcgctcctc caagccagtt acctcggttc aaagagttgg tagctcagag aaccttcgaa 240aaaccgccct gcaaggcggt tttttcgttt tcagagcaag agattacgcg cagaccaaaa 300cgatctcaag aagatcatct tattaatcag ataaaatatt tctagatttc agtgcaattt 360atctcttcaa atgtagcacc tgaagtcagc cccatacgat ataagttgta attctcatgt 420tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg 480gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc 540ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg 600ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc 660aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg 720gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag 780ctgtcttcgg tatcgtcgta tcccactacc gagatgtccg caccaacgcg cagcccggac 840tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg 900ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag 960tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca 1020gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg 1080tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata 1140atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag 1200gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg 1260acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct 1320accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca 1380atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt 1440ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct 1500tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg 1560gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc 1620accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc 1680cattcgatgg tgtccgggat ctcgacgctc tcccttacta gtttgacaat taatcatcgg 1740ctcgtataat gtgtggaatt gtgagcggat aacaattaag gaggttccga tgaatcatgc 1800ttcagattgc acctgtgaag agagtctgtg tgaaacgcta cgcgcgtttt ccgctcagca 1860tcccgatagc gtgctgtatc aaacttcgct gatgagcgcc ctgctcagcg gcgtctacga 1920aggtaccacc accattgcgg acctgctgaa gcacggtgat ttcgggctcg gcacttttaa 1980tgaactcgac ggcgagctga tcgcgtttag cagccaggtt tatcaactgc gtgccgacgg 2040cagcgcgcgt aaagcgcgtc cggaacagaa aacgccgttt gcggtgatga cctggtttca 2100gccgcagtac cgtaaaacct ttgaccatcc ggtcagccgc cagcagctgc atgaggttat 2160tgaccagcaa attccttccg acaatctgtt ctgcgcgctg cgaatcgatg gtcatttccg 2220ccacgcccat acccgcaccg tgcctcgtca gacgccgccc taccgggcga tgaccgacgt 2280gctcgacgat cagccggttt tccgctttaa ccagcgtgac ggcgtactgg tcggttttcg 2340taccccgcag catatgcagg gaattaacgt cgccggctat cacgaacact tcattaccga 2400tgaccgccag ggcggcggcc acctgctgga ctaccagctc gaccatgggg tattgacctt 2460cggcgaaatt cataagctga tgatcgacct tcccgccgac agcgcgttcc tgcaggccaa 2520tttgcatccc gataatctcg atgccgccat ccgttcagta gaaagttagg aggttcacat 2580ggacaaacag tatccgcagc gccagtgggc gcacggcgcc gatctggtcg tcagccaact 2640ggaagcgcaa ggcgtacggc aggtcttcgg gatccccggc gctaaaatcg ataaggtttt 2700cgactcgttg ctggactcct caatccgcat tattccggta cgtcacgagg ccaacgccgc 2760ctttatggcc gccgcggtcg ggcgcattac cggcaaagcg ggcgtcgcgc tggtgacctc 2820cggacccggt tgttccaacc tgataaccgg gatggccacc gccaatagcg aaggcgaccc 2880ggtggtggcg ctgggcggcg cggtcaaacg cgcggataaa gccaaacagg tacaccagag 2940tatggacacg gtggcgatgt tcagcccggt caccaaatac gcggtagaag tgacctcgcc 3000ggatgcgctg gcggaagtgg tttctaacgc ttttcgcgcc gccgagcagg gtcgcccggg 3060cagcgccttc gtcagtctgc cgcaggatgt ggtcgatggt ccggtgaccg gcaaagtcct 3120gcccgccagc agcgcgccgc agatgggcgc cgcgcctgac gaggcaatca atcaggttgc 3180gaagttgatt gcccaggcga agaatccggt gttcctgctt ggattaatgg ccagccagac 3240ggaaaacagc gccgcgctgc atcgtttgct ggaaaccagc catattccgg tcaccagcac 3300ctatcaggcc gccggggcgg tcaatcagga taacttctcg cgcttcgccg ggcgcgtcgg 3360gctgtttaac aatcaggccg gtgaccgctt attgcaactg gccgacctgg ttatctgcat 3420cggctatagc ccggtggaat acgaaccggc gatgtggaac agcggcaacg cgacgctggt 3480acatatcgac gtactgcccg cctatgaaga gcgtaactac acgccggatg tcgagctggt 3540gggcgacatc gccggcacgc tgaacaagct ggcgcaaaat atcgatcatc ggctggtgct 3600ctcgccgcag gctgctgaaa tcctccacga ccgccagcat cagcgggaac tgcttgaccg 3660ccgcggagcg cagttgaatc agtttgccct gcacccgctg cgtatcgttc gcgccatgca 3720ggatatcgtc aacagcgacg tcacgctgac ggtcgatatg gggagcttcc atatctggat 3780cgcccgctat ctctacagct tccgcgcccg tcaggtgatg atctccaacg gtcagcagac 3840catgggcgtc gccctgccgt gggccatcgg ggcctggctg gtcaatccgc agcgcaaagt 3900ggtctcggtc tccggcgatg gcggttttct gcaatccagc atggagctgg aaacggcggt 3960ccgcctgaaa gccaacatcc tgcatcttat ctgggtcgat aacggctaca acatggtcgc 4020catccaggaa gagaaaaaat atcaacgcct gtccggcgtc gagttcggtc ctatggattt 4080taaagcctat gccgaatcct tcggcgcgaa agggtttgcg gtggaaagcg ctgaggcgct 4140ggagccgacg ctacgcgcgg cgatggacgt cgacggcccg gcggtggtcg ccatccccgt 4200ggattaccgt gataacccgc tgctgatggg ccagctacac ctgagtcaaa ttctttaagt 4260catcacaaaa ggaaatggaa atgaaaaaag tcgcacttgt caccggcgcc ggtcagggca 4320ttggtaaagc tatcgcgtta cgcctcgtga aggacggttt tgccgtggcg atcgccgatt 4380acaatgacgt cacagcgaaa gccgtggcgg atgaaatcaa ccagcacggc ggccgggcaa 4440tcgcggtcaa agtcgatgtt tccgaccgtg agcaggtgtt tgccgccgtc gaacaggcgc 4500gaaaaacgct gggcggattc aacgtcatcg tcaataacgc cggggtcgcg ccatcaacgc 4560ctatcgaatc cattacgccg gagattgtcg acaaggtcta caacatcaac gttaaagggg 4620tgatctgggg gattcaggcg gcagtcgagg cctttaaaaa agaggggcac ggcggcaaaa 4680tcatcaacgc ctgttcgcag gccggacacg tcggcaaccc ggaactggcg gtctacagct 4740cgagcaaatt cgccgtacgc ggtttaacgc aaaccgccgc tcgcgacctg gcgccgctgg 4800gtattaccgt taacggctac tgcccgggga ttgtgaaaac gccgatgtgg gccgagatcg 4860atcgtcaggt atccgaagcg gcgggtaaac ctctgggcta cgggacagcc gaattcgcca 4920aacgcatcac cctcggccgc ctgtctgagc cagaagatgt cgccgcctgc gtctcttatc 4980tcgccagccc ggattccgat tatatgaccg gtcaatcgct gctgatcgat ggcgggatgg 5040tattcaatta attaacctag gctgctgcca ccgctgagca ataactagca taaccccttg 5100gggcctctaa acgggtcttg aggggttttt tgctgaaacc tcaggcattt gagaagcaca 5160cggtcacact gcttccggta gtcaataaac cggtaaacca gcaatagaca taagcggcta 5220tttaacgacc ctgccctgaa ccgacgaccg ggtcgaattt gctttcgaat ttctgccatt 5280catccgctta ttatcactta ttcaggcgta gcaccaggcg tttaagggca ccaataactg 5340ccttaaaaaa attacgcccc gccctgccac tcatcgcagt actgttgtaa ttcattaagc 5400attctgccga catggaagcc atcacagacg gcatgatgaa cctgaatcgc cagcggcatc 5460agcaccttgt cgccttgcgt ataatatttg cccatagtga aaacgggggc gaagaagttg 5520tccatattgg ccacgtttaa atcaaaactg gtgaaactca cccagggatt ggctgagacg 5580aaaaacatat tctcaataaa ccctttaggg aaataggcca ggttttcacc gtaacacgcc 5640acatcttgcg aatatatgtg tagaaactgc cggaaatcgt cgtggtattc actccagagc 5700gatgaaaacg tttcagtttg ctcatggaaa acggtgtaac aagggtgaac actatcccat 5760atcaccagct caccgtcttt cattgccata cggaactccg gatgagcatt catcaggcgg 5820gcaagaatgt gaataaaggc cggataaaac ttgtgcttat ttttctttac ggtctttaaa 5880aaggccgtaa tatccagctg aacggtctgg ttataggtac attgagcaac tgactgaaat 5940gcctcaaaat gttctttacg atgccattgg gatatatcaa cggtggtata tccagtgatt 6000tttttctcca ttttagcttc cttagctcct gaaaatctcg ataactcaaa aaatacgccc 6060ggtagtgatc ttatttcatt atggtgaaag ttggaacctc ttacgtgccg atcaacgtct 6120cattttcgcc aaaagttggc ccagggcttc ccggtatcaa cagggacacc aggatttatt 6180tattctgcga agtgatcttc cgtcacaggt atttattcgg cgcaaagtgc gtcgggtgat 6240gctgccaact tactgattta gtgtatgatg gtgtttttga ggtgctccag tggcttctgt 6300ttctatcagc tgtccctcct gttcagctac tgacggggtg gtgcgtaacg gcaaaagcac 6360cgccggacat cagcgctagc ggagtgtata ctggcttact atgttggcac tgatgagggt 6420gtcagtgaag tgcttcatgt ggcaggagaa aaaaggctgc accggtgcgt cagcagaata 6480tgtgatacag gatatattcc gcttcctcgc tcactgactc gctacgctcg gtcgttcgac 6540tgcggcgagc ggaaatggct tacgaacggg gcggagattt cctggaagat gccaggaaga 6600tacttaacag ggaagtgaga gggccgcggc aaagccgttt ttccataggc tccgcccccc 6660tgacaagcat cacgaaatct gacgctcaaa tcagtggtgg cgaaacccga caggactata 6720aagataccag gcgtttcccc tggcggctcc ctcgtgcgct ctcctgttcc tgcctttcgg 6780tttaccggtg tcattccgct gttatggccg cgtttgtctc attccacgcc tgacactcag 6840ttccgggtag gcagttcgct ccaagctgga ctgta 68755629DNAArtificial SequencePromoter_gamma 56ttgacaatta atcatcggct cgtataatg 295735DNAArtificial SequenceSynthetic 5' UTR_gamma 57ggaattgtga gcggataaca attaaggaga tatgc 355835DNAArtificial SequencePromoter_FLP 58ttgacggcta gctcagtcct aggtacagtg ctagc 355925DNAArtificial SequenceSynthetic 5' UTR_FLP 59atctcgcaaa tcgaaggagc ctcat 256035DNAArtificial SequencePromoter_crtE 60ttgacggcta gctcagtcct aggtacagtg ctagc 356125DNAArtificial SequenceSynthetic 5' UTR_crtE 61cacgaccata taaggaggat agaag 256235DNAArtificial SequencePromoter_idi 62ttgacggcta gctcagtcct aggtacagtg ctagc 356325DNAArtificial SequenceSynthetic 5' UTR_idi 63catcaacaaa taaggaggag ctaca 256435DNAArtificial SequencePromoter_ispA 64ttgacggcta gctcagtcct aggtacagtg ctagc 356525DNAArtificial SequenceSynthetic 5' UTR_ispA 65cacgaccata taaggaggat agaag 256635DNAArtificial SequencePromoter_dxs 66ttgacggcta gctcagtcct aggtacagtg ctagc 356725DNAArtificial SequenceSynthetic 5' UTR_dxs 67cgtctccaca taaggagcag ttcac 256829DNAArtificial SequencePromoter_BudA 68ttgacaatta atcatcggct cgtataatg 296938DNAArtificial SequenceSynthetic 5' UTR_BudA 69tgtggaattg tgagcggata acaattaagg aggttccg 38

Claims

1. A Vibrio sp. DHG strain having a high-performance metabolic pathway for utilization of carbon sources, wherein the stain has an accession number KCTC13239BP.

2. The Vibrio sp. DHG strain of claim 1, wherein the carbon source includes at least one selected from the group consisting of glucose, mannitol, sucrose, arabinose, galactose, glycerol, xylose, mannose, fructose, lactose, maltose, sucrose, alginic acid, cellulose, dextrin, glycogen, hyaluronic acid, lentinan, Zymosan, chitosan, glucan, lignin and pectin.

3. The Vibrio sp. DHG strain of claim 1, wherein the strain comprises a 16S rDNA gene represented by a nucleotide sequence of SEQ ID NO: 1.

4. The Vibrio sp. DHG strain of claim 1, wherein the strain comprises a beta gene represented by a nucleotide sequence of SEQ ID NO: 2 or a beta protein represented by an amino acid sequence of SEQ ID NO: 3.

5. The Vibrio sp. DHG strain of claim 1, wherein the strain comprises an exo gene represented by a nucleotide sequence of SEQ ID NO: 4 or an exo protein represented by an amino acid sequence of SEQ ID NO: 5.

6. A transformed Vibrio sp. DHG strain obtained by introducing a gene encoding a gamma protein represented by a nucleotide sequence of SEQ ID NO: 6 into the Vibrio sp. DHG strain of claim 1.

7. A transformed strain for lycopene production, wherein a crtEBI gene represented by a nucleotide sequence of SEQ ID NO: 9 is further introduced thereinto.

8. The transformed strain for lycopene production of claim 7, wherein an idi gene represented by a nucleotide sequence of SEQ ID NO: 10 is further introduced thereinto.

9. The transformed strain for lycopene production of claim 7, wherein an ispA gene represented by a nucleotide sequence of SEQ ID NO: 11 is further introduced thereinto.

10. The transformed strain for lycopene production of claim 7, wherein a dxs gene represented by a nucleotide sequence of SEQ ID NO: 12 is further introduced thereinto.

11. A transformed strain for producing 2,3-butanediol, wherein the transformed strain is obtained by introducing, into the transformed Vibrio sp. DHG strain of claim 6, at least one gene selected from the group consisting of: a budA gene represented by a nucleotide sequence of SEQ ID NO: 13; a budB gene represented by a nucleotide sequence of SEQ ID NO: 14; and a budC gene represented by a nucleotide sequence of SEQ ID NO: 15.

12. The transformed strain for producing 2,3-butanediol of claim 11, wherein the transformed strain is obtained by deleting, from the transformed Vibrio sp. DHG strain of claim 6, at least one gene selected from the group consisting of: a IdhA gene represented by a nucleotide sequence of SEQ ID NO: 16; a frdA gene represented by a nucleotide sequence of SEQ ID NO: 17; a frdB gene represented by a nucleotide sequence of SEQ ID NO: 18; a frdC gene represented by a nucleotide sequence of SEQ ID NO: 19; a frdD gene represented by a nucleotide sequence of SEQ ID NO: 20; a pflB gene represented by a nucleotide sequence of SEQ ID NO: 21.

13. A lycopene production method comprising culturing the transformed strain for lycopene production of claim 7.

14. A method for producing 2,3-butanediol, the method comprising culturing the transformed strain for producing 2,3-butanediol of claim 11.

15. A SXT recombination system expression cassette comprising: a synthetic 5' UTR (untranslated region); a promoter represented by one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 22 to 35 and 56; and at least one gene selected from the group consisting of a gene encoding a beta protein, a gene encoding an exo protein, and a gene encoding a gamma protein.

16. The SXT recombination system expression cassette of claim 15, wherein the synthetic 5' UTR is represented by a nucleotide sequence of SEQ ID NO: 57.

17. A recombinant vector comprising the SXT recombination system expression cassette of claim 15.

18. A flippase gene expression cassette, comprising: a synthetic 5' UTR (untranslated region); a promoter represented by one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 22 to 35 and 58; and a gene encoding a flippase.

19. The flippase gene expression cassette of claim 18, wherein the synthetic 5' UTR is represented by a nucleotide sequence of SEQ ID NO: 59.

20. A recombinant vector comprising the flippase gene expression cassette of claim 18.

21. The crtEBI gene expression cassette, comprising a synthetic 5' UTR (untranslated region); a promoter represented by one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 22 to 35 and 60; and a crtEBI gene.

22. The crtEBI gene expression cassette of claim 21, wherein the synthetic 5' UTR is represented by a nucleotide sequence of SEQ ID NO: 61.

23. The crtEBI gene expression cassette of claim 21, wherein the crtEBI gene expression cassette further comprises an idi gene expression cassette.

24. The crtEBI gene expression cassette of claim 23, wherein the idi gene expression cassette comprises: a synthetic 5' UTR (untranslated region) represented by a nucleotide sequence of SEQ ID NO: 63; a promoter represented by one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 22 to 35 and 63; and an idi gene.

25. The crtEBI gene expression cassette of claim 21, wherein the crtEBI gene expression cassette further comprises an ispA gene expression cassette.

26. The crtEBI gene expression cassette of claim 25, wherein the ispA gene expression cassette comprises: a synthetic 5' UTR (untranslated region) represented by a nucleotide sequence of SEQ ID NO: 65; a promoter represented by one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 22 to 35 and 64; and an ispA gene.

27. The crtEBI gene expression cassette of claim 21, wherein the crtEBI gene expression cassette further comprises a dxs gene expression cassette.

28. The crtEBI gene expression cassette of claim 27, wherein the dxs gene expression cassette comprises: a synthetic 5' UTR (untranslated region) represented by a nucleotide sequence of SEQ ID NO: 67; a promoter represented by one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 22 to 35 and 66; and a dxs gene.

29. A recombinant vector comprising the crtEBI gene expression cassette of claim 21.

30. A budACB operon expression cassette, comprising: a synthetic 5' UTR (untranslated region); a promoter represented by one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 22 to 35 and 68; and one or more genes selected from the group consisting of a gene encoding a BudA protein, a gene encoding a BudC protein, and a gene encoding a BudB protein.

31. The budACB operon expression cassette of claim 30, wherein the synthetic 5' UTR is represented by a nucleotide sequence of SEQ ID NO: 69.

32. A recombinant vector comprising the budACB operon expression cassette of claim 30.